Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.     

Canine morphology in the larger Felidae: implications for feeding ecology

Canine morphology is analysed at seven intervals along the crown in both anteroposterior and lateromedial perspective in seven species of large felids. The puma and the snow leopard have stout, rather conical canines, whereas those of lions, jaguars, and tigers bear substantial resemblance to each other, reflecting their phylogenetic relationships, and are less conical and large. The canines of the leopard are intermediate in morphology between those of the other species, probably reflecting its more generalized diet. The clouded leopard has very large and blade-like canines, which are different from the other analysed species. Canine bending strengths to estimated bite forces appear to differs less among the species than morphology, indicating that the evolution of canines has been constricted with respect to their strength in failure, probably owing to their being equally important for species fitness. However, the clouded leopard again stands out, having a high estimated bite force and rather weak canines in bending about the anteroposterior as well as lateromedial planes compared to the other species. Canine morphology to some extent reflects differences in killing mode, but also appears to be related to the phylogeny. The marked divergence of the clouded leopard is presently not understood.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 573–592.     

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca)

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.     

Using PGFM (13,14-dihydro-15-keto-prostaglandin F2α) as a non-invasive pregnancy marker for felids

Understanding the complex endocrine interactions that control reproduction in felids is essential for captive breeding management. The most important demand is a quick and reliable pregnancy diagnosis. However, the occurrence of pseudopregnancies in felids complicates matters. We investigated whether the fecal prostaglandin metabolite (PGFM) recently suggested for pregnancy diagnosis in the lynx is suitable for all felid species. We found that increased levels of PGFM during the last trimester indicate pregnancy in seven of the eight main lineages of the carnivore family Felidae. PGFM levels in a sand cat (domestic cat lineage) were basal at mating and remained so until Day 40 post-mating. Day 41 marked the beginning of a distinct increase culminating in peak levels of 6.5 μg/g before parturition and decreasing again to baseline thereafter. Similar pregnancy profiles were obtained from the domestic cat, the leopard cat, the lynx, the ocelot and the caracal lineage, whereas in pseudopregnant individuals (sand cat, Iberian and Eurasian lynx) fecal PGFM remained at basal levels. In pregnant cheetahs (puma lineage) PGFM increased above basal following day ∼48 peaking before pregnancy but remained at baseline in pseudopregnant females. Discrepancies existed in the Panthera lineage. While Chinese leopard, Sumatran tiger, and the black panther showed marked increases of PGFM during the last weeks of pregnancy, only moderate increases in PGFM levels were found in the Indochinese tiger and the Persian leopard. Altogether, PGFM as tool for pregnancy diagnosis has been proven to be useful in breeding management of felids.     

Lynx reproduction – Long-lasting life cycle of corpora lutea in a feline species

A review of lynxes’ reproductive biology and comparison between the reproductive cycles of the domestic cat and lynxes is presented. Three of the four lynx species (the bobcat excluded) express quite similar reproductive pattern (age at sexual maturity, estrus and pregnancy length, litter size). Similarly to the domestic cat, the bobcat is polyestric and can have more than one litter per year. Domestic cats and many other felid species are known to express anovulatory, pregnant and pseudo-pregnant reproductive cycles in dependence on ovulation induction and fertilization. The formation of corpora lutea (CLs) occurs after ovulation. In pregnant animals, luteal function ends with parturition, whereas during pseudo-pregnancy a shorter life span and lower hormone secretion are observed. The life cycle of corpora lutea in Eurasian lynxes is different from the pattern described in domestic cats. Lynx CLs produce progestagens in distinctive amounts permanently for at least two years, regardless of their origin (pregnancy or pseudo-pregnancy). It is suggested that long-lasting CLs induce a negative feedback to inactivate folliculogenesis, turning a normally polyestric cycle observed in most felids into a monoestric cycle in lynxes.     

动物园大型猫科动物犬瘟热的免疫监测

虎、狮、豹等大型猫科动物可发生犬瘟热(CD),有必要进行预防。采取某动物园未免疫的东北虎、非洲狮、猞猁血样,用中和试验检测,结果显示这些动物的犬瘟热病毒(CDV)抗体水平均小于1:5。用CD弱毒疫苗接种以上动物,幼龄动物免疫14d逐步产生抗体;成年动物体内抗体水平在两月内迅速升高,并维持在1:64以上,9～11个月后逐步下降,加强免疫后抗体又迅速升高至≥1:112。该疫苗对所有接种的动物均安全可靠。白化的孟加拉虎接种疫苗后不能产生CDV中和抗体。     

动物园大型猫科动物犬瘟热的免疫监测

虎、狮、豹等大型猫科动物可发生犬瘟热(CD),有必要进行预防.采取某动物园未免疫的东北虎、非洲狮、猞猁血样,用中和试验检测,结果显示这些动物的犬瘟热病毒(CDV)抗体水平均小于15.用CD弱毒疫苗接种以上动物,幼龄动物免疫14d逐步产生抗体;成年动物体内抗体水平在两月内迅速升高,并维持在164以上,9～11个月后逐步下降,加强免疫后抗体又迅速升高至≥1112.该疫苗对所有接种的动物均安全可靠.白化的孟加拉虎接种疫苗后不能产生CDV中和抗体.     

A molecular view of pinniped relationships with particular emphasis on the true seals

Phylogenetic analysis of conservative nucleotide substitutions in 18 complete sequences of the mitochondrial cytochrome gene of Phocidae (true seals), Odobenidae (walruses), and Otariidae (sea lions and fur seals), plus three ursid and three felid sequences, identified the pinnipeds as monophyletic with Otariidae and Odobenidae on a common evolutionary branch. Analysis of total nucleotide differences separated the evolutionary lineages of northern and southern phocids. Both lineages are distinct from the most ancestral phocid genus, Monachus (monk seals), represented by the Hawaiian monk seal. The inclusion of the Hawaiian monk seal in the subfamily Monachinae makes the subfamily paraphyletic. Among the northern phocids, the hooded seal (genus Cystophora, chromosome number 2n = 34) is sister taxon to the Phoca complex. The Phoca complex, which is characterized by the chromosome number 2n = 32, includes genus Phoca and the monotypic genus Halichoerus (grey seal). The comparison does not support a generic distinction of Halichoerus within the Phoca complex. The present data suggest that Cystophora and Phoca separated 6 million years ago. Among the southern phocids the close molecular relationship of the Weddell and leopard seals relative to their morphological distinction exemplifies rapid adaptation to different ecological niches. This result stands in contrast to the limited morphological differentiation relative to the pronounced molecular distinctions that may occur within the Phoca complex.Correspondence to: Ú. Árnason     

PCR-CTPP: a rapid and reliable genotyping technique based on ZFX/ZFY alleles for sex identification of tiger (<Emphasis Type="Italic">Panthera tigris</Emphasis>) and four other endangered felids

An inexpensive, time-saving and reliable method, polymerase chain reaction with confronting two-pair primers (PCR-CTPP), was developed for sex identification in tiger (Panthera tigris) based on zinc finger alleles (ZFX/ZFY). A site of “C/G” transversion representing fixed differences that discriminated between ZFX and ZFY exons among felids was identified for primers designing. This primer set was successfully tested on samples including blood, shed hairs, dried skin, and stool which contained potential contamination caused by prey DNA. Cross species tests shown that this primer set was also useful for sex identification in four other endangered felids.