Feline Sperm Research Articles

Sperm ubiquitination in epididymal feline semen

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.

Ejaculation training, seminal alkaline phosphatase and semen preservation through cooling in a milk-based extender in domestic cats.

The purpose of this report is to describe (1) the training of domestic cats in ejaculation into an artificial vagina (AV), (2) alkaline phosphatase (AP) concentrations in whole ejaculates, and (3) the in vitro effect of a skimmed-milk plus egg yolk (SM-Y) extender on feline spermatozoa incubated at 4ºC. Five post-pubertal cats were trained to ejaculate into an AV three times a week for 20 mins in the presence of a teaser queen. Fifty AV-obtained ejaculates were macro- and microscopically assessed, and the AP therein measured by optimized colorimetry. Eighty AV-obtained ejaculates were pooled, diluted in SM-Y extender [80% (v/v) skimmed milk, 20% (v/v) egg yolk, and antibiotics], stored at 4°C and evaluated daily for 6 days. All the animals could be trained to ejaculate, although the interval up to the first AV ejaculation varied from 1.5 to 5.5 months (mean 3.9 months). The final performance at collection ranged from excellent to poor and was inversely related to the training period required in all cases. The mean AP concentration in whole ejaculates was 20,645.6 ± 4405U/l, which was not correlated with the concentration of spermatozoa. Most seminal parameters [(%); total (77 ± 2.3) and progressive (62.7 ± 3.4) motility, live sperm (91.8 ± 1.2), intact plasmalemma (83.5 ± 2.6), normal acrosomes (83.5 ± 2.6), pH (6.6 ± 0.0) and osmolarity (mOsm/l; 321 ± 5.2)], though decreasing during storage in the cold, remained within values compatible with in vivo fertilization for 2 days.

Effect of Different Extenders onIn VitroCharacteristics of Feline Epididymal Sperm During Cryopreservation

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS-egg yolk-based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25-ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0- and 6-h post-thaw. Experiment 1 compared the effect of three non-feline commercial extenders - based on TRIS-egg yolk (Triladyl), egg-yolk-free medium (AndroMed) and skimmed milk-egg yolk (Gent) - on the quality of frozen-thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p<0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen-thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS-egg yolk-based extenders, one non-feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing-thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing-thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS-egg yolk-based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.

69 AN EASY-TO-PERFORM METHOD TO ASSESS VIABILITY OF FELINE FREEZE-DRIED SPERM

Freeze-drying sperm seems to be the ultimate alternative to preserve sperm cells from endangered species because of its easiness in transportation and storage of samples. This technique has already been used for equine, bovine, murine, rabbit, canine, and feline sperm cells. However, it is still important to verify the DNA integrity of such samples to produce viable offspring. The objective of this study was to verify the reliability of acridine orange (AO) staining to assess DNA damage of feline freeze-dried sperm samples. Three normospermic cats were used as sperm donors, and sperm samples were collected with the use of an artificial vagina without the presence of an oestrous queen. The control group (G1) used only fresh semen. To increase the range of sperm variability before freeze-drying, the sperm samples were supplemented with 2 extenders, namely SOF (G2) and human tubal fluid (G3). These extenders were selected because of their lack of cryoprotectant agents with a view to producing membrane damage and thus putting DNA integrity at risk. Samples were taken to a freeze-dryer (Edwards do Brasil, Brazil) to produce stable free-dried sperm. Over 50 samples were assessed for DNA damage by using AO and alkaline comet assay (CA). Acridine orange taints red/orange the sperm cells that present sperm damage, and CA shows long comet tails for cells with DNA damage. The CA was selected to countercheck the AO results owing to its higher accuracy. Nevertheless, both AO and CA count 100 cells per analysis, and a total of 3 repetitions per sample were sufficient. For the statistical analysis, SAS PROC GLM was used, and the significance level was set at 0.05. The AO staining showed that the G1 samples had a greater DNA damage (27.1%) if compared with G2 (15.2%) and G3 (23.1%; P &lt; 0.0001). Nevertheless, G2 and G3 displayed sufficient DNA integrity throughout the whole lyophilization process. The CA, which is a more precise evaluation technique, proved that the groups had distinguished results but still were ordered in the same way: G1 was the one with more DNA damage (17.7%), G3 was second (14.4%), and G2 was third (3.2%) in DNA alterations. Just like AO, the CA showed difference among treatments (P &lt; 0.0001). Since AO is a technique that requires very little sophistication and presented about the same results as the very elaborate CA method, it might be used on a daily basis to assess feline sperm DNA that underwent the freeze-drying process.

Spermatogenic Function in Cats

There are limited data on feline sperm production. We exhausted epididymal spermatozoa (i.e. the number of ejaculated spermatozoa <5 × 10(6)) 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 × 10(6).

Distribution of Glycoproteins on Feline Testicular Sperm, Epididymal Sperm and Ejaculated Sperm

Glycoproteins (GPs) are known to be involved in the phenomenon of sperm maturation and capacitation. In the present study, we investigated the attachment of GPs on sperm cell membrane during the process of feline sperm maturation from testicular sperm to ejaculated sperm by using 8 FITC-labeled lectins. The results showed that 3 types of GPs were presented on testicular sperm and 7 on caput epididymal sperm. Corpus and cauda epididymal sperm and ejaculated sperm had GPs detected by 8 FITC-labeled lectins used in the present study. This study demonstrates the part of the characteristic of GPs that are present on the feline sperm cell membrane during the process of sperm maturation.

240 FUNCTIONAL TRAITS OF CAT SPERM DURING DISTINCT MATURATION STATUS

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P &lt; 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.

95 THE EFFECT OF FREEZING TECHNIQUES ON QUALITY OF CAT TESTICULAR SPERM

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 × 5 mm) and cryopreserved in freezing medium containing with 5% (v/v) glycerol using conventional (10 min 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 &gt; 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.

Cryopreservation of feline epididymal spermatozoa from dead and alive animals and its use in assisted reproduction

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.

Functional Role of Feline Zona Pellucida Protein 4 Trefoil Domain: A Sperm Receptor or Structural Component of the Domestic Cat Zona Pellucida?

The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function.

MTT（3[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide）を用いた犬および猫精子の生存性検査

MTT（3[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide）を用いた犬および猫精子の生存性検査

Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides

The aim of this study was evaluate the survival ability of canine and feline spermatozoa maintained within epididymides stored at 4 °C for 24, 48 or 72 h in sterile isotonic saline solution (SAL) or a Tris–egg yolk (TEY) storage medium. Fifteen domestic dogs and 15 cats were neutered and their testes were placed in TEY or SAL and stored at 4 °C for either 24, 48 or 72 h. Sperm samples were obtained by cutting the cauda epididymides into a Tris extender and were evaluated for motility, velocity, viability, plasma membrane integrity, and acrosome morphology. In dogs, there were no significant differences between storage media for motility, plasma membrane integrity, viability and velocity. However, dog sperm stored in TEY had better acrosome morphology compared to sperm stored in SAL ( P < 0.05). Dog sperm recovered at 72 h had a reduction in all parameters studied compared to those recovered at 24 h ( P < 0.05). In cats, sperm recovered from epididymides stored in TEY had higher motility, plasma membrane integrity and velocity at all times compared to those stored in SAL ( P < 0.05). Cat sperm recovered at 72 h had reduced motility, acrosome morphology, viability and velocity compared to those recovered at 24 h ( P < 0.05). The addition of TEY to canine epididymal sperm, thus, had a better protective effect than SAL only on acrosome morphology. In cats, in contrast, TEY had a better protective effect than SAL on all epididymal sperm parameters studied. In both species, sperm recovered at 72 h had a significant reduction in all parameters studied compared to those recovered at 24 h.

Osmotic Effects on Feline Spermatozoa from Normospermic versus Teratospermic Donors

Effects of osmolality stresses on the sperm of normospermic (>60% normal sperm/ejaculate) versus teratospermic (<40% normal sperm) domestic cats and the normospermic leopard cat and the teratospermic clouded leopard were studied. Spermatozoa were exposed to various anisotonic solutions in a single step or returned to near isotonic conditions in a single step after exposure to anisotonic solutions. The percentage of sperm motility was measured subjectively, and dual fluorescent stains were used to assess membrane integrity by flow cytometry. The percentage of sperm motility declined (P < 0.05) in domestic cat sperm exposed to osmolalities <200 and >450 mOsm. Spermatozoa from all felines underwent marked (P < 0.05) membrane disruption following a hypotonic stress, but sperm from teratospermic donors experienced greater (P < 0.05) membrane disruption in response to decreased osmolality. While feline spermatozoa appeared to be highly resistant to hypertonic (600, 1200, and 2400 mOsm) conditions, with >85% of the cells maintaining intact membranes, severe membrane disruption occurred when cells were returned to isotonicity in a single step. There was no difference (P > 0.05) between a 1- and 5-min exposure to various anisotonic solutions. Similarly, sperm from normospermic and teratospermic domestic cats responded identically after exposure to ionic or nonionic solute. Results demonstrate that: (1) spermatozoa from teratospermic males are more vulnerable to a hypotonic stress than sperm from normospermic counterparts; (2) in response to small deviations in osmolality, feline sperm experience a more rapid decline in motility than membrane integrity; and (3) an abrupt return to isotonicity after a hypertonic stress causes extensive sperm membrane damage regardless of ejaculate quality.

Ovarian and testicular function in the domestic cat: clinical management of spontaneous reproductive disease

Abstract Vaginal cornification and/or serum progesterone concentration may be used as indicators of follicular and luteal function in the cat in diagnosis of estrus, induced or spontaneous ovulation, ovarian remnant syndrome, and prolonged gestation. Feline sperm may be collected by electroejaculation, ejaculation into an artificial vagina, cystocentesis of the male following ejaculation or by vaginal wash of the bred female. Normal feline seminal plasma contains high concentrations of alkaline phosphatase, and may contain more than 10 000 colony forming units (CFU) ml−1 bacteria, most of which are normal preputial flora. Serum testosterone concentration, often undetectable (less than 0.02 ng ml−1) in the normal male, increases 1 h after gonadotropin-releasing hormone (GnRH) or 4 h after human chorionic gonadotropin (hCG) administration in the tom. Presence of penile spines is an indicator of testicular tissue or testosterone administration. Gonadectomy performed at 7 weeks or 7 months of age in male and female cats is associated with increased length of long bones due to delayed physeal closure, decreased heat production, and increased risk of obesity, and not with decrease in urethral diameter. Normal penile extrusion was not possible in 6 6 male cats neutered at 7 weeks of age or 2 5 male cats neutered at 7 months of age, owing to persistence of the balanopreputial fold, which normally undergoes dissolution under the influence of androgens.