Nuclear and microtubule remodeling and in vitro development of nuclear transferred cat oocytes with skin fibroblasts of the domestic cat (Felis silvestris catus) and leopard cat (Prionailurus bengalensis)

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is a threatened animal in South Korea. In terms of protecting endangered felids, nuclear transfer (NT) is a potentially valuable technique for assuring the continuation of species with dwindling numbers. In the present experiment, nuclear and microtubule remodeling and the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with nuclei of somatic cells from either domestic cat fibroblast (DCF) or leopard cat fibroblast (LCF) were evaluated. Microtubule aster is allocated to de-condensed chromatin following nuclear transfer (3h after activation) of fibroblast cells from both domestic and leopard cats, suggesting the introduction of a somatic cell centrosome. The transferred fibroblast nuclei formed a large, swollen, pronuclear-like structure in most reconstructed oocytes, in the cat or leopard cat. At 18h following nuclear transfer, mitosis occurred, and according to the photo (F) it appears that spindle microtubules and two asters were observed. The percentages of blastocyst formation from nuclear transfer embryos derived from domestic cat fibroblasts (4/46, 8.6%) were not significantly different than those for nuclear transfer embryos constructed with leopard cat fibroblasts (4/52, 7.6%). These results indicate that nuclear and microtubule remodeling processes and in vitro developmental ability are similar in reconstructed cat oocytes following transfer of nuclei from either domestic or leopard cats.     

In vitro fertilization of gonadotropin-stimulated leopard cat (Felis bengalensis) follicular oocytes

Based on techniques developed for the domestic cat, in vitro fertilization (IVF) studies were conducted in the taxonomically related leopard cat (Felis bengalensis). Adult females received pregnant mares' serum gonadotropin (PMSG) followed 80 or 84 h later by human chorionic gonadotropin (hCG) on two to four occasions over a 40-day to 27-month interval. Oocytes were collected laparoscopically from ovarian follicles 25-27 h after hCG and co-cultured with processed, homologous spermatozoa (37 degrees C, 5% CO2 in air, humidified atmosphere) for 30-36 h. There was no apparent ovarian refractoriness to repeated treatments with exogenous gonadotropins. Overall, the mean number of mature follicles present and the total number of oocytes and proportion of immature oocytes collected did not differ (P greater than 0.05) between the 80 h (4.9 +/- 0.9; 4.7 +/- 1.2; 14.9%, respectively) and 84 h (5.6 +/- 1.4; 5.4 +/- 1.7; 22.2%, respectively) gonadotropin interval groups. However, the proportion of mature leopard cat oocytes fertilized in vitro, as determined by embryonic cleavage, was increased (P less than 0.005) by extending the interval between PMSG and hCG from 80 (17.5%) to 84 (52.4%) h. These data 1) demonstrate that, compared to the domestic cat, the ovaries of the leopard cat are less responsive to a given PMSG/hCG treatment; 2) indicate that leopard cat follicular oocytes can be recovered readily by laparoscopy and are capable of becoming fertilized in vitro; and 3) suggest that IVF may be a viable approach for producing embryos and perhaps enhancing captive propagation of rare Felidae.     

Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay.

The ability of domestic cat or leopard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt-stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recovered after ovariectomy and matured in vitro for 18-36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4 degrees C, 0.5-24 weeks) in a HEPES-buffered hypertonic salt solution. Electroejaculated, washed sperm (2-4 x 10(6) sperm/ml) were preincubated for 1.0 h (38 degrees C, 5% CO2 in air) and then co-incubated (2 x 10(5) sperm/ml) with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein-free, modified Tyrode's solution (TLP-PVA) or in the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP-PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P greater than 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 +/- 5.4%; leopard cat, 38.6 +/- 2.8%) or stored ZP (domestic cat, 32.4 +/- 4.2%; leopard cat, 27.6 +/- 2.3%). Sperm incubated in protein-free medium (TLP-PVA) were less capable (P less than 0.05) of ZP penetration (domestic cat, 14.6 +/- 5.9%; leopard cat, 7.9 +/- 3.0%) than sperm incubated in medium TALP-PVA containing BSA (domestic cat, 60.3 +/- 5.9%; leopard cat, 58.4 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ecology driving genetic variation: a comparative phylogeography of jungle cat (Felis chaus) and leopard cat (Prionailurus bengalensis) in India

Background

Comparative phylogeography links historical population processes to current/ecological processes through congruent/incongruent patterns of genetic variation among species/lineages. Despite high biodiversity, India lacks a phylogeographic paradigm due to limited comparative studies. We compared the phylogenetic patterns of Indian populations of jungle cat (Felis chaus) and leopard cat (Prionailurus bengalensis). Given similarities in their distribution within India, evolutionary histories, body size and habits, congruent patterns of genetic variation were expected.

Methodology/Principal Findings

We collected scats from various biogeographic zones in India and analyzed mtDNA from 55 jungle cats (460 bp NADH5, 141 bp cytochrome b) and 40 leopard cats (362 bp NADH5, 202 bp cytochrome b). Jungle cats revealed high genetic variation, relatively low population structure and demographic expansion around the mid-Pleistocene. In contrast, leopard cats revealed lower genetic variation and high population structure with a F _ST of 0.86 between North and South Indian populations. Niche-model analyses using two approaches (BIOCLIM and MaxEnt) support absence of leopard cats from Central India, indicating a climate associated barrier. We hypothesize that high summer temperatures limit leopard cat distribution and that a rise in temperature in the peninsular region of India during the LGM caused the split in leopard cat population in India.

Conclusions/Significance

Our results indicate that ecological variables describing a species range can predict genetic patterns. Our study has also resolved the confusion over the distribution of the leopard cat in India. The reciprocally monophyletic island population in the South mandates conservation attention.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.     

Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.     

When domestic cat (Felis silvestris catus) population structures interact with their viruses

Many theoretical studies have proposed different causal mechanisms by which the structure of a host population could have important implications for life history traits of pathogens. However, little information is available from real systems to test these hypotheses. The domestic cat, Felis silvestris catus, whose populations exhibit a great variability in social and spatial structure, represent an ideal case study to assess this question. In the present article, we show how cat population structure may have influenced the evolution of feline viruses and, in return, how these viruses may have modified the genetic structure of cat populations. To cite this article: D. Pontier et al., C. R. Biologies 332 (2009).     

A cytogenetic study of subfertility in the domestic cat (Felis catus)

The chromosomes of cat embryos derived from three subfertile queens were studied 4 weeks postcoitum. The incidence of spontaneous chromosome anomalies was 15% and type of abnormality observed was autosomal mosaicism. The significance of this finding and the developmental fate of the chromosomally aberrant embryos is discussed. The absence of polyploidy and (nonmosaic) autosomal trisomy, which are the most common observations in previous studies on mammalian embryos, suggests that the difference between the present observations and previous studies may be related to the difference in gestational stage studied, or to the difference in ovulatory patterns between domestic cats and other mammals. Polyploidy through ageing of the gametes is less likely in the cat than in spontaneous ovulators since ovulation in cats is induced only after coitus. It is speculated that chromosome analysis of earlier stages of embryonic development may reveal more severe forms of karyotype alteration.     

The primary structure of hemoglobins from the domestic cat (Felis catus, Felidae)

The complete primary structure of the two hemoglobin components of the domestic cat (Felis catus) is presented. The major component (A) accounts for 60-70% whereas the minor component (B) constitutes 30-40% of the total hemoglobin. Separation of the polypeptides was carried out in buffers containing 8M urea on CM-Cellulose. The sequence was studied by Edman degradation of tryptic and cyanogen bromide cleavage products in a liquid phase sequencer. The sequence is compared for homology with human hemoglobin. The beta-chain of the minor components (beta B) has a blocked N-terminal residue identified as acetylserine whereas that of the major component (beta A) is free glycine. The two hemoglobins have identical alpha-chains and differ with respect to their beta-chains at the following positions (beta B/beta A): beta NA1 Ac-Ser/Gly, beta A1 Ser/Thr, beta H17 Ser/Asn and beta HC1 Arg/Lys. The structural and functional aspects of these exchanges are discussed.     

A genetic linkage map of microsatellites in the domestic cat (Felis catus)

Of the nonprimate mammalian species with developing comparative gene maps, the feline gene map (Felis catus, Order Carnivora, 2N = 38) displays the highest level of syntenic conservation with humans, with as few as 10 translocation exchanges discriminating the human and feline genome organization. To extend this model, a genetic linkage map of microsatellite loci in the feline genome has been constructed including 246 autosomal and 7 X-linked loci. Two hundred thirty-five dinucleotide (dC. dA)n. (dG. dT)n and 18 tetranucleotide repeat loci were identified and genotyped in a two-family, 108-member multigeneration interspecies backcross pedigree between the domestic cat (F. catus) and the Asian leopard cat (Prionailurus bengalensis). Two hundred twenty-nine loci were linked to at least one other marker with a lod score >/=3.0, identifying 34 linkage groups. Representative markers from each linkage group were assigned to specific cat chromosomes by somatic cell hybrid analysis, resulting in chromosomal assignments to 16 of the 19 feline chromosomes. Genome coverage spans approximately 2900 cM, and we estimate a genetic length for the sex-averaged map as 3300 cM. The map has an average intragroup intermarker spacing of 11 cM and provides a valuable resource for mapping phenotypic variation in the species and relating it to gene maps of other mammals, including human.     

Tyrosinase mutations associated with Siamese and Burmese patterns in the domestic cat (Felis catus)

The Siamese cat has a highly recognized coat colour phenotype that expresses pigment at the extremities of the body, such as the ears, tail and paws. This temperature-sensitive colouration causes a 'mask' on the face and the phenotype is commonly referred to as 'pointed'. Burmese is an allelic variant that is less temperature-sensitive, producing more pigment throughout the torso than Siamese. Tyrosinase (TYR) mutations have been suspected to cause these phenotypes because mutations in TYR are associated with similar phenotypes in other species. Linkage and synteny mapping in the cat has indirectly supported TYR as the causative gene for these feline phenotypes. TYR mutations associated with Siamese and Burmese phenotypes are described herein. Over 200 cats were analysed, representing 12 breeds as well as randomly bred cats. The SNP associated with the Siamese phenotype is an exon 2 G > A transition changing glycine to arginine (G302R). The SNP associated with the Burmese phenotype is an exon 1 G > T transversion changing glycine to tryptophan (G227W). The G302R mutation segregated concordantly within a pedigree of Himalayan (pointed) Persians. All cats that had 'pointed' or the Burmese coat colour phenotype were homozygous for the corresponding mutations, respectively, suggesting that these phenotypes are a result of the identified mutations or unidentified mutations that are in linkage disequilibrium. Because the same mutations were identified in different breeds with similar phenotypes, the mutations are likely to be identical by descent rather than multiple mutation events occurring at the same site.     

The social function of tail up in the domestic cat (Felis silvestris catus)

We investigated the social function of tail up in order to verify its possible relationship with the hierarchical organization of a social group. Domestic cats live at higher densities than their ancestor which is a solitary species. Since the signals needed by solitary animals have different properties than those needed by group-living individuals, signalling pattern utilised by the domestic cat has inevitably changed. Kittens displayed the tail up when greeting their mother; this behaviour can also be observed in wild species. But, in domestic cat the tail up can be also observed when an adult individual meets another one and it signals the intention to interact amicably. Rank order affected the display of tail up posture: it was more frequently displayed by low-ranking cats, and high-ranking individuals received it more often than other members of the social group. Then, tail up seems to be a signal by means of which a cat shows the recognition of the higher social status of the individual to whom is directed. We confirmed the association between tail up and other affiliative behavioural patterns and the individual variability in displaying them. Considerations on the evolution of the tail up as a visual signal will be discussed.     

Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation

Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus-oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco's modified Eagle medium (DMEM) alone as a control group or supplemented with 100 ng/ml IGF-I. After culturing for 32-34 h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.     

A set of microsatellite markers for population genetics of leopard cat (Prionailurus bengalensis) and cross-species amplification in other felids

We describe the isolation and characterization of novel microsatellite loci from the leopard cat, Prionailurus bengalensis Kerr, 1792 (Family Felidae). Using Illumina HiSeq2500 sequencing technology, we sequenced the leopard cat genome and identified 1.5 million loci of simple sequence repeats with di- to deca-nucleotide motifs. We developed twelve polymorphic markers with tetra-nucleotide motif types after screening 35 loci for amplification and polymorphism. The observed and expected heterozygosities of the markers were 0.438 and 0.423, respectively. The number of alleles per locus ranged from 2 to 7, with a mean polymorphism information content of 0.383. Eleven loci were at Hardy-Weinberg equilibrium and no linkage disequilibrium was detected among any pairs of loci. We tested cross-species amplification of these markers across five other felids (Panthera tigris, P. pardus, P. onca, Acinonyx jubatus, and Felis catus). All loci were transferable to at least one other feline species and four amplified all five species. The microsatellite markers developed in this study will be valuable for estimating ecological parameters of populations and to establish conservation and management strategies for feline species.     

First successful transfer of cryopreserved feline (Felis catus) embryos resulting in live offspring

Sexually mature domestic cats were hormonally stimulated to induce superovulation; embryo recovery was accomplished by laparotomy. Embryos were frozen by conventional embryo freezing methods used in the domestic cattle embryo transfer industry. Thawing was achieved in a 28 degrees C or 37 degrees C waterbath or in ambient air. Overnight culture of the frozen-thawed embryos in a supplemented Nutrient Mixture F-10 (Ham's) or Earle's Balanced Salt Solution with 20% heat-treated newborn calf serum resulted in five successful term litters from recipient queens. Embryo recipients who became pregnant had been treated with a subcutaneous injection of follicle-stimulating hormone (FSH-P) once every 24 hr for 6 days in the amount of 0.2 mg/day for the first 5 days and 0.1 mg on the sixth day, followed by two intramuscular 750 IU injections of human chorionic gonadotropin 24 hr apart, beginning on the same day as the sixth injection of FSH-P.     

Production of nuclear transfer llama (lama glama) embryos from in vitro matured llama oocytes

To date, there have been no reports of somatic cell nuclear transfer in llamas. The application of this methodology to the camelid industry could be helpful in the propagation of genetically valuable animals. The objective of this study was to produce nuclear transfer llama embryos comparing the development of these llama embryos cultured in either CR1aa medium (treatment A) or G1.2 medium (treatment B) medium. Llamas were superstimulated by double dominant follicle reduction 12 days apart, followed by pFSH administered in daily descending doses over a 3-day interval (total dose of 200 mg). Animals were ovariectomized by flank laparotomy, follicles were aspirated from excised ovaries and oocytes were in vitro matured for a 30-h period. Adult female llama fibroblasts were used as donor karyoplasts and injected into enucleated llama oocytes. Embryo development was assessed after 2 days of culture. A total of 307 follicles were aspirated from nine treated females, resulting in 298 (97%) oocytes recovered. Of a total of 229 evaluated oocytes, 120 (52%) achieved nuclear maturation. Of a total of 80 reconstructed couplets, 50 (62.5%) were successfully fused. Subsequent cleavage rates were 32 and 40% for treatments A and B, respectively, with no significant difference (p < 0.05) detected between treatment groups. A total of 11 embryos (8-cell to morula stages) were transferred to synchronized recipient llamas. Ultrasonography at 14 days post-transfer indicated that no pregnancies were established. This study shows that nuclear transfer can be successfully applied to the production of llama embryos. Further research is needed to identify optimal parameters to improve efficiency of nuclear transfer in this species.     

基于红外相机数据评估华南地区豹猫的种群密度和活动节律

可靠的种群密度数据对野生动物的保护和管理十分重要。豹猫(Prionailurus bengalensis)是中国分布最广且常见的猫科动物, 但野生种群密度估算的研究并不多。本研究于2020年6月至2021年5月在香港新界嘉道理农场暨植物园开展红外相机调查, 利用空间标记-重捕法估算当地豹猫的种群密度并用核密度估计方法分析其活动节律。本次调查以网格方式布置红外相机, 在约1.5 km²的研究范围之内设置了19个相机位点, 每个位点安装2台相机以获取豹猫身体两侧花纹来进行个体识别。连续12个月调查共捕获113次有效的豹猫拍摄事件, 当中仅61次事件的照片足够清晰以进行个体识别。基于种群封闭的要求, 我们以2个月为单位将12个月的数据分为6个采样期去分析豹猫种群密度, 结果显示仅两个采样期的估算值最为准确, 分别为0.64 ± 0.31 (0.26-1.55)只/km²和0.87 ± 0.48 (0.31-2.40)只/km², 是已知全球豹猫密度最高的地点之一。结果还发现, 雨季研究地点的豹猫并无明显的日活动节律, 在旱季则偏夜行-晨昏行性多一些, 但也有一定的日间活动; 雨季和旱季的日活动节律无显著差异。本研究是首次以个体识别配以空间标记-重捕模型对中国大陆地区豹猫种群密度调查的研究; 我们也提出一些关于红外相机架设方法的建议, 以提高照片个体识别的准确度并增加重捕次数, 最后提高密度估算的准确度。本研究也进一步证明豹猫适应性极强, 在活动节律上表现出极高的可塑性, 在严格保护下可以恢复健康的种群。     

Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage.

Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25 degrees C (no cooling; control), 2) cooled to 5 degrees C in a commercial refrigerator for 30 min (rapid cooling; approximately 4 degrees C/min), 3) placed in an ice slush at 0 degrees C for 10 min (ultrarapid cooling; approximately 14 degrees C/min), or 4) cooled to 0 degrees C at 0.5 degrees C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4 degrees C. All samples then were warmed to 25 degrees C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25 degrees C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling ( approximately 4 degrees C/min) to 5 degrees C (N, 65.6%; T, 27.5%) or ultrarapid cooling ( approximately 14 degrees C/min) to 0 degrees C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5 degrees C/min) to 5 degrees C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0 degrees C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.     

Observations on variability in LH release and fertility during oestrus in the domestic cat (Felis catus)

Hormonal changes, behaviour, ovulation and fertility were examined in response to coitus at two different times during oestrus in the female domestic cat housed in conditions of natural light (N = 13). On Day 2 or Day 4/5 of oestrus females were allowed 1 copulation in 15 min (single matings) or 2-3 copulations in 30 min (multiple matings). Plasma LH, oestradiol-17 beta and progesterone concentrations during the 24-h period after coitus were measured by radioimmunoassay; ovulation was assumed to have occurred if progesterone values were elevated 7-30 days after coitus. With the exception of 2 out of 3 animals receiving single matings on Day 2 of oestrus, all animals showed subsequent elevated progesterone values. Females receiving multiple matings had significantly greater releases of LH as measured by the area under the curve than those receiving single matings. There was significantly greater variability in the LH response of queens on Day 2 of oestrus compared to those on Day 4/5 for peak values and area under the curve; the only failure in release of LH was in queens on Day 2. Oestradiol levels did not differ significantly between Day 2 and Day 4/5 of oestrus. Progesterone values remained less than 1 ng/ml for 24 h after coitus. Both LH peak values and area under the curve were significantly greater for animals that became pregnant. There were also significant differences in coital behaviour between queens on Day 2 and those on Day 4/5 of oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)