Equine chorionic gonadotropin induces in vitro follicular growth from the multi-layered secondary developmental stage in cats.

Equine chorionic gonadotropin (eCG) has been commonly used to induce estrus in several felid species. However, the mechanisms by which this gonadotropin regulates cat folliculogenesis are still unclear. We investigated the responsiveness of cat ovarian follicles at different developmental stages to various eCG concentrations supplemented in vitro. Follicles were mechanically isolated from the ovaries of 22 cats and categorized into three developmental stages based on their morphology and diameter: 1) two-layered secondary follicle (SF), 100-150 µm (n = 139); 2) multi-layered SF, 150-300 µm (n = 154); and 3) early antral follicle (AF), ≥300-500 µm (n = 123). The follicles were then encapsulated in 0.5% (w/v) sodium alginate and cultured for 12 days in Minimum Essential Medium supplemented with 0, 0.05, 0.1 or 0.5 IU/mL eCG. Follicle and oocyte diameters were assessed every 3 days. On Day 12, mRNA expression levels of FHSR, LHCGR, GDF9, BMP15, CYP17A1, CYP19A1 and STAR were analyzed using real-time PCR. After being cultured for 12 days, follicle growth and mRNA expression of two-layered SF were not influenced by eCG at all concentrations (P > 0.05). However, the concentration of eCG at 0.05 IU/mL stimulated follicular growth and gene expressions in the multi-layered SF and early AF (P < 0.05). Correspondingly, the diameter of oocytes in the multi-layered SF and early AF treated with 0.05 IU/mL eCG was unchanged. Considering the mRNA expression, the level of STAR was enhanced in the early AF (P < 0.05) and tended to increase in the multi-layered SF (P = 0.08) cultured in 0.05 IU/mL eCG, whereas the expression of other genes was not affected. In sum, the responsiveness of cat follicles to eCG is apparent from the multi-layered SF stage onward. The eCG supplementation at 0.05 IU/mL appeared to be optimal for the follicle culture in the domestic cats.

The expression of kisspeptin and its receptor in the domestic cat ovary and uterus in different stages of the ovarian cycle.

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P < 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis.

Dynamic changes in mitochondrial DNA, distribution and activity within cat oocytes during folliculogenesis.

Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial (n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral (n = 44), early antral (n = 66), small antral (n = 59), antral (n = 41) and advanced antral (n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mtDNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.

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 7 days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7 days 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 2 hr after a jungle cat (Felis chaus) aged 10 years died during anaesthesia plus another necropsy-to-sperm recovery time of 25 hr 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.

Mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (Neofelis nebulosa).

Clouded leopards (Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n = 9) were allocated to simple washing (control; resulting in 11.7% ± 1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9% ± 0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution (HOS, 60 mOsm fructose solution) containing 5 mM dithiothreitol (DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 (TX) (a detergent) was added to the HOS/DTT-treated samples. After HOS/DTT incubation, the control samples and sperm-selected samples presented 73.4% ± 3.1% and 73.9% ± 2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8% ± 3.8% and 26.5% ± 3.4%; p = .1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6% ± 10.4% and 21.1% ± 7.9%; p = .06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation.

Beneficial effect of extracellular adenosine 5'-triphosphate treatment on the Indochinese leopard (Panthera pardus delacouri) sperm quality after cryopreservation.

The Indochinese leopard (Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization (IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5'-triphosphate (ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 106 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process (p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ (p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced (p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) (p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased (p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development).

Influence of living status (single vs. paired) and centrifugation with colloids on the sperm morphology and functionality in the clouded leopard (Neofelis nebulosa).

The objectives of the present study were to evaluate sperm characteristic of captive clouded leopards in Thailand and examine the structural and functional properties of sperm after selection with the single-layer centrifugation (SLC) method. Twenty-two ejaculates from 11 captive clouded leopards (four housed with access to a female in estrus, and seven housed singly) were collected and assessed for semen traits during 2013 to 2015. Twelve fresh ejaculates were chosen from seven males, and each was divided between two sperm preparation methods; (1) simple washing and (2) SLC. Cryopreservation was performed after semen preparation. Sperm qualities after selections including motility, progressive motility, sperm motility index, viability, acrosome integrity, DNA integrity, and morphology were evaluated in fresh, chilled, and frozen-thawed samples. In addition, sperm functionality after cryopreservation was tested by heterologous IVF using domestic cat oocytes. Sperm motility in the ejaculates was 52.5% to 91.3% (76.8 ± 2.0%, mean ± standard error). A high proportion of morphologically abnormal sperm (63.9 ± 2.0%) was observed, with the major abnormality being tightly coiled tail (13.5 ± 0.5%). An interesting observation was that males housed together with a female had a significantly higher proportion of sperm with intact acrosome (47.9 ± 3.4% and 38.4 ± 2.8%) and lower proportion of sperm with bent midpiece and droplet (7.1 ± 0.6% and 10.2 ± 0.5%) than the males living singly. The sperm motility index, intact acrosome, and sperm with normal tail in the fresh and chilled semen samples were improved by the SLC. In the postthawed semen, the SLC selected higher numbers of viable sperm (34.1 ± 2.2% and 27.9 ± 1.8%), sperm with intact acrosome (31.2 ± 2.1% and 24.3 ± 2.2%), and sperm with normal tail (34.2 ± 2.7% and 24.3 ± 2.7%) than simple washing. Also, the proportion of sperm with tightly coiled tail was lower in the SLC-processed than the simple washed samples (8.1 ± 3.1% and 13.5 ± 3.4%). The SLC-processed group had significantly higher penetration rate in heterologous IVF (29.4 ± 3.0%) than the simple washing group (15.8 ± 3.2%). In conclusion, ejaculates of clouded leopards living in Thailand demonstrated teratospermic characteristic similar to the previous reports from other continents. Single-layer centrifugation is a promising tool to select morphologically normal sperm of teratospermic donors. The successes of assisted reproductive technology could be enhanced by the improved quality of postthaw sperm in this species.

Chelation of trace elements in preservation medium influences the quality of boar spermatozoa during liquid preservation at 5°C for 4 weeks.

BACKGROUND: The addition of a metal chelator, ethylenediaminetetraacetic acid (EDTA), to semen extender has the purpose of capturing trace element ions. OBJECTIVE: This study was conducted to evaluate the effects of EDTA on the quality and in vitro fertilisability of liquid-preserved boar spermatozoa. METHODS: In Experiment 1, semen samples were preserved in the semen extender supplemented with 0, 3, 6, or 12 mM of Na-EDTA at 5 degree C for 4 weeks. In Experiment 2, semen samples were preserved in the extender supplemented with 3 mM of Na-EDTA, Ca-EDTA, or Zn-EDTA and without chelator EDTA. RESULTS: When Na-EDTA was used as a chelating substance in the extender, 3 mM was a most suitable concentration for sperm motility and viability after cold preservation. The supplementation of 3 mM Ca-EDTA had advantages regarding sperm motility, viability and plasma membrane integrity. CONCLUSION: Our findings indicate that 3 mM Ca-EDTA is the most suitable metal-chelating substance for the liquid preservation of boar semen.

Cell cycle synchronization of skin fibroblast cells in four species of family Felidae.

This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 µm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 µm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.

Effects of cold storage prior to freezing on superoxide dismutase, glutathione peroxidase activities, level of total reactive oxygen species and sperm quality in dogs.

Reactive oxygen species (ROS) are one of several crucial factors that cause mammalian sperm damage during handling and preservation. Their deleterious effects can be restricted by the action of antioxidants. The present study aimed at investigating: (i) effects of cold storage prior to freezing on activities of enzymatic antioxidants (superoxide dismutase; SOD and glutathione peroxidase; GPx) and level of total reactive oxygen species (tROS); and (ii) effects of SOD or SOD plus GPx supplementation to chilled (3 and 96 h) and frozen-thawed semen. Six privately owned dogs were included. Experiment I: Each pooled semen was divided into three equal aliquots, which were subjected to chilled storage for 3, 24 or 96 h prior to freezing (n = 7). The activities of SOD and GPx in sperm cells and tROS level in chilled and frozen-thawed semen were measured. Experiment II: Pooled semen was divided to be cold stored for 3 or 96 h in three different extenders; (i) Uppsala Equex extender (control), (ii) Uppsala Equex extender supplemented SOD, or (iii) Uppsala Equex extender supplemented with SOD plus GPx. Sperm motility, viability and integrity of acrosome and DNA was evaluated after cold storage and frozen-thawed. The cold storage from 24 h prior to freezing resulted in a decrease in the SOD activity in the frozen-thawed sperm cells whereas the GPx activity and tROS levels were not affected. In addition, the supplementation of SOD plus GPx enhanced the percentage of sperm viability and DNA integrity after cold stored and frozen-thawed. In sum, the SOD activity is compromised by cold storage prior to freezing of dog semen. Addition of GPx is suggested to assist SOD to complete the enzymatic ROS scavenging system in the dog sperm.

Cryopreservation of cat testicular tissues: effects of storage temperature, freezing protocols and cryoprotective agents.

Cryopreservation of testicular tissue has become a part of gamete preservation in wild animal post-mortem. Using domestic cats as a model for wild felids, this study aimed to (i) investigate the effect of temperature for testicular tissue storage on sperm quality; (ii) compare efficiency of freezing protocols; and (iii) evaluate properties of cryoprotective agents to protect testicular sperm quality. A pair of testes from each cat (n = 9) was cut into four pieces. Three randomly selected pieces were allocated to be (i) fresh controls; (ii) stored at 4 °C for 24 h; and (iii) stored at room temperature (28 °C) for 24 h. After storage, the testicular tissue from each group was cut into 10 small pieces. One piece was assigned to be a control while the others were assigned to three freezing protocols; -80 °C (n = 3), vitrification (n = 3) or two-step freezing (kept above liquid nitrogen vapour for 10 min and submerged in liquid nitrogen) (n = 3). Each of three pieces was frozen using dimethyl sulphoxide (DMSO), ethylene glycol (EG) or DMSO combined with EG. Sperm membrane (SYBR-14/EthD-1) and DNA (acridine orange) integrity were evaluated before and after cryopreservation. The storage of testicular tissue at room temperature decreased the percentage of sperm with intact membrane in fresh tissue (59.5 ± 30.5 vs 87.9 ± 7.0%, p < 0.05). DNA integrity was decreased after 24-h storage either at 4 °C or room temperature (p < 0.05). The two-step freezing resulted in a higher percentage of sperm with intact plasma membrane than the other techniques. Dimethyl sulphoxide, EG and DMSO combined with EG provided similar protection for the sperm membrane and DNA from cryodamages. In conclusion, storage of testicular tissue at 4 °C is necessary to maintain sperm membrane integrity during transportation of tissue for cryopreservation in the freezing laboratory. The results provide information for male gamete rescue in felid particularly when they die unexpectedly in the field where freezing facilities are not well equipped.

The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps).

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm.

The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, approximately 200 x 10(6) sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300 x g for 20 min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean+/-SD of 16.4+/-8.7, 10.7+/-8.9, and 2.3+/-1.7%, respectively; P<0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02+/-0.01, 0.02+/-0.04, 0.03+/-0.04, and 0.44+/-0.22 x 10(6) cells/mL, respectively; P<0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P>0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P<0.05), due to the lowest proportion of coiled tails (P<0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P>0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up.

Intergeneric somatic cell nucleus transfer in marbled cat and flat-headed cat.

Intergeneric nucleus transfer (ig-NT) is a promising technique to produce offspring of endangered species. The objectives of this study were to (1) investigate the in vitro development of marbled cat (MC; Pardofelis marmorata) and flat-headed cat (FC; Prionailurus planiceps) ig-NT embryos reconstructed from domestic cat (DC; Felis catus) oocytes (Experiment 1), (2) evaluate the effect of individual FC donor cell lines on NT success (Experiment 2), and (3) assess the developmental ability of FC-cloned and DC-IVF embryos in vitro and in vivo after oviductal transfer (Experiment 3). In Experiment 1, the morula rate of FC-reconstructed embryos was significantly higher than those of MC and DC embryos but lower than that of parthenogenic DC embryos. However, blastocyst rate was not different. In Experiment 2, FC-ig-NT embryos reconstructed from female muscular tissue had significantly higher morula rate in comparison with those derived from other donor cell lines. However, there was no difference in blastocyst rate among cell lines. In Experiment 3, in vitro development of FC-ig-NT embryos was lower than that of DC-IVF embryos. The competency of in vivo development of FC-ig-NT and/or DC-IVF embryos was investigated by assessing pregnancy rate after their transfer into DC recipients. Domestic cat recipients receiving only FC-ig-NT embryos, FC-ig-NT embryos in one side of the oviduct and DC-IVF embryos contralaterally (co-transfer), and only DC-IVF embryos were observed. No pregnancy was detected in all recipients receiving FC-ig-NT embryos. One recipient receiving co-transferred embryos became pregnant, then delivered DC-IVF dead fetuses (n=2) and live kittens (n=6). All recipients receiving DC-IVF embryos became pregnant, and three of six recipients delivered five DC-IVF kittens. These results illustrate the developmental capacity of MC- and FC-ig-NT embryos up to the blastocyst stage. Individual donor cell line affects the developmental success up to the morula stage of FC-ig-NT embryos. Improving the developmental competence and quality of FC-ig-NT embryos may be required for implantation and development to term of FC-ig-NT offspring.

Effects of thawing temperature and post-thaw dilution on the quality of cat spermatozoa.

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70 degrees 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 degrees C for 15 s, (ii) at 37 degrees C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70 degrees C for 6 s, (iv) at 70 degrees 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 degrees C and post-thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post-thaw incubation.

Effects of the anti-progestin aglepristone on the uterine tissue of cats administered medroxyprogesterone acetate.

The effect of aglepristone, a progesterone receptor antagonist, on feline endometrial proliferation induced by medroxyprogesterone acetate (MPA), a contraceptive progestin, was investigated. Three groups each of six cats were studied: Group A, during inter-oestrus; Group B, given 50 mg MPA; Group C, receiving 50 mg MPA followed by 10 mg/kg aglepristone twice at 24-h interval 3 weeks later. Ovariohysterectomy was performed, and uterine tissue was collected from the mid part of the horn when the cats were confirmed as being in inter-oestrus (Group A), 3 weeks after the MPA administration (Group B), or 2 weeks after aglepristone administration (Group C). Uterine weights, relative uterine weights and uterine diameters were determined. The endometrial and myometrial thicknesses and the endometrial/myometrial ratios were determined in histological sections. Histological sections were also immunostained for proliferative activity [anti-proliferative cell nuclear antigen (PCNA) antibody], and progesterone receptor (anti-hPR) and percentage of positive cells in luminal vs glandular epithelium were determined for both parameters. PCNA and all morphological parameters except endometrial thickness were increased (p < 0.05) by MPA, and progesterone receptor was decreased compared to untreated cats. Aglepristone treatment had no significant effect compared to MPA alone. However, glandular proliferative activity was usually but not significantly lower in Group C than in Group B, suggesting a potential effect of the anti-progestin that might be clinically beneficial. Alternatively, this possible effect in the aglepristone group may reflect the longer time after MPA that these animals were examined. Whether lack of significant effect of the anti-progestin was because of too high dose of MPA relative to the dose of aglepristone, the delay of 14 days in obtaining tissues after aglepristone treatment, or simply an insufficient duration of aglepristone treatment requires further study.

Detection of lipid peroxidation reaction in frozen-thawed epididymal cat spermatozoa using BODIPY(581/591) C11.

Lipid peroxidation is considered to be an important cause of sperm membrane damage, resulting in an apparent reduction of reproductive fecundity. Recently, a new lipophilic fluorescent dye probe (BODIPY(581/591) C11; Invitrogen Singapore Pte Ltd, Singapore) has been demonstrated to be a highly sensitive indicator for the physiological oxidation of cell membrane fatty acids. The objectives of this study were: (i) to detect lipid peroxidation in frozen-thawed epididymal cat spermatozoa using the BODIPY(581/591) C11 and (ii) to study the effect of semen extender in protecting sperm membrane from lipid peroxidation [100-mm ferrous ion, ferrous sulphate (FeSO(4))]. Epididymal cat spermatozoa were collected from eight male cats. Two straws of sperm sample from each cat were cryopreserved. After thawing, the semen extender from the first straw was removed and the sperm pellet was resuspended with Tris buffer (control). The semen sample from the other straw was equally divided: one sample contained semen extender (treatment A) and one contained no extender (treatment B); both were incubated with FeSO(4). Semen samples were labelled with the BODIPY(581/591) C11 probe and evaluated by flow cytometry at 0 and 6 h after thawing (control), 6 h after the addition of FeSO(4) (treatment A), and 30 min and 6 h after the addition of FeSO(4) (treatment B), respectively. The percentage of lipid peroxidation was higher after treatment B (51.3 +/- 23.9) and 6-h incubation compared with the control and treatment A (p < 0.05). Furthermore, the percentage of lipid peroxidation after treatment B increased during the incubation time (p < 0.05). In conclusion, the high percentage of lipid peroxidation after treatment B indicated that FeSO(4) induced membrane damage in cat spermatozoa, which could be detected by BODIPY(581/591) C11. This marker is suggested to be a beneficial tool for the evaluation of lipid peroxidation. Furthermore, the use of semen extender seemed to protect cat spermatozoa membranes from lipid peroxidation.

Survival of frozen-thawed cat spermatozoa pre-cooled in the epididymides.

Sperm recovery from epididymides and their cryo-preservation require particular media and equipment not always available in the field. This study aimed to compare the quality of frozen-thawed epididymal cat spermatozoa pre-cooled in an extender vs. cooled in the epididymides. Testes with attached epididymides from 23 cats subjected to routine orchidectomy were allocated into two groups; 2-day (n = 10) and 4-day cold storage (n = 13). Spermatozoa from one epididymis of each pair were recovered, evaluated, extended in a Tris egg yolk extender, cooled to 5 degrees C and stored for either 2 or 4 days prior to freezing (Treatment 1). The remaining testis attached with the epididymis was stored at 5 degrees C in a Tris-buffered solution for either 2 or 4 days, before the spermatozoa were harvested, evaluated and frozen (Treatment 2). The spermatozoa were evaluated for percentage of motility, viability, intact acrosomal integrity and morphology. Sperm evaluations were performed prior to freezing and after freezing-thawing. The spermatozoa maintained in the epididymes yielded similar quality after cold storage and after freeze-thawing in comparison to that maintained in the extender at both storage times (p > 0.05). However, cold storage of spermatozoa for 4 days resulted in a reduction in motility compared to that cold stored for 2 days (p < 0.05). Similarly, freezing of spermatozoa pre-cooled for 4 days resulted in lower percentages of motility, viability and intact acrosome than that pre-cooled for 2 days (p < 0.05). A higher number of spermatozoa with simple bent tail and coiled tail were found in the samples maintained in the epididymides than in the extender after 4-day storage. The results indicated that cat spermatozoa can be cold stored in the epididymides prior to being harvested and cryopreserved in a distant laboratory. This technology is an alternative to rescue male gamete when animals are castrated or unexpectedly die under field conditions.

Incubation of post-thaw epididymal cat spermatozoa with seminal plasma.

In domestic cats, epididymal spermatozoa have lower initial motility and viability than ejaculated spermatozoa and it is possible that seminal plasma compounds are behind these effects. The aim of this study was to investigate whether co-incubation of post-thaw epididymal cat spermatozoa with seminal plasma was able to improve sperm quality. Epididymal cat spermatozoa from 11 cats were cryopreserved. After thawing, each sperm sample was divided into two aliquots, centrifuged and incubated with two different media; Tris buffer (control) or pooled seminal plasma (treatment). Sperm quality was observed at 0, 2, 4 and 6 h after incubation. The results demonstrated that all of the sperm parameters except acrosome integrity were lower in the treatment group compared to the control group (p < 0.05); the percentages of motility (46.4 +/- 15.4 vs 40.0 +/- 9.4), the scores of progressive motility (3.1 +/- 0.4 vs 2.8 +/- 0.5), the percentages of spermatozoa with intact plasma membrane (46.3 +/- 9.7 vs 39.6 +/- 8.9) and intact acrosome (36.5 +/- 16.2 vs 32.9 +/- 15.1), as well as at all time points. In conclusion, the seminal plasma seems less beneficial to the post-thaw epididymal cat spermatozoa than the Tris buffer.