JoGayle Howard

Genetic Restoration of the Florida Panther

Return of the Florida Panther The Florida panther is an endangered lineage that has been the target of extensive human management to maintain genetic diversity within a small population. Now, Johnson et al. (p. 1641; see the Perspective by Packer) provide comprehensive genetic and demographic analyses of a project to rescue the Florida panther from inbreeding, low genetic diversity, inbreeding depression, and demographic stochasticity by outcrossing it to Texas pumas. The genetics and reproductive fitness traits of different panther populations and their crosses reveal the benefits of outcrossing on survival, genetic diversity, male reproductive characteristics, and population demography. Recovery of the Florida panther population is attributed to the benefits of admixture with panthers from Texas. The rediscovery of remnant Florida panthers (Puma concolor coryi) in southern Florida swamplands prompted a program to protect and stabilize the population. In 1995, conservation managers translocated eight female pumas (P. c. stanleyana) from Texas to increase depleted genetic diversity, improve population numbers, and reverse indications of inbreeding depression. We have assessed the demographic, population-genetic, and biomedical consequences of this restoration experiment and show that panther numbers increased threefold, genetic heterozygosity doubled, survival and fitness measures improved, and inbreeding correlates declined significantly. Although these results are encouraging, continued habitat loss, persistent inbreeding, infectious agents, and possible habitat saturation pose new dilemmas. This intensive management program illustrates the challenges of maintaining populations of large predators worldwide.

Embryogenesis in conservation biology — Or, how to make an endangered species embryo

Abstract Embryo technologies have not as yet contributed to practical conservation of rare wildlife species. Production of young following artificial insemination (AI), embryo transfer (ET) or in vitro fertilization (IVF) has been sporadic, and it is now clear that biological differences among species limit our abilities to adapt these techniques easily to rare species. Nonetheless, there is encouraging progress at two levels. First, there is more acceptance that rare wildlife species safely tolerate the manipulations necessary to collect basic reproductive information or to test artificial breeding. This has increased access to rare animal populations and helped develop organized captive breeding programs, many of which emphasize the need for more research. Secondly, a gradually developing database about how these species reproduce is driving more systematic experimentation and artificial breeding attempts. Studies in our laboratory focus on producing embryos in vivo or in vitro. When essential information is available on fundamental reproductive processes, and, especially when comparative data are available from a domesticated animal model, then AI techniques are adapted to the endangered species. When few data are available, then studies emphasize using IVF (often in combination with in vitro oocyte maturation) to examine the factors regulating embryo formation and viability. These strategies are illustrated by recent progress involving (i) AI of select species of cervids, felids and mustelids, (ii) oocyte maturation in felids and (iii) IVF and ET in felids. Offspring have been produced, but perhaps more important are the answers to fundamental and mechanistic questions about why some wildlife species thrive and others do not. If reality-based conservation is defined as a continual data-gathering process that assimilates any and all information ultimately useful for preserving species, then embryo technologies are making considerable contributions to conservation biology.

REPRODUCTIVE CHARACTERISTICS OF MALE FLORIDA PANTHERS: COMPARATIVE STUDIES FROM FLORIDA, TEXAS, COLORADO, LATIN AMERICA, AND NORTH AMERICAN ZOOS

Testicular volume, semen traits, and pituitary-gonadal hormones were measured in populations of Felis concolor from Florida, Texas, Colorado, Latin America, and North American zoos. More Florida panthers ( F. concolor coryi ) were unilaterally cryptorchid (one testicle not descended into the scrotum) than other populations (43.8 versus 3.9%, respectively). Florida panthers also had lower testicular and semen volumes, poorer sperm progressive motility, and more morphologically abnormal sperm, including a higher incidence of acrosomal defects and abnormal mitochondrial sheaths. Transmission electron microscopy revealed discontinuities in the acrosome, extraneous acrosomal material under the plasma membrane, and remnants of the golgi complex under the acrosome. No differences were detected in mean-circulating follicle-stimulating hormone, luteinizing hormone, or testosterone between Florida panthers and other populations of mountain lions. Seminal traits and concentrations of follicle-stimulating hormone, luteinizing hormone, and testosterone were similar between cryptorchid and noncryptorchid Florida panthers. Animals with F. concolor coryi ancestry were categorized on the basis of amount of genetic variation (low = type A; medium = type B; high = captive Piper stock). Compared to counterparts, type A Florida panthers had the lowest testicular volume and sperm-motility ratings and were the only animals exhibiting unilateral cryptorchidism. These results demonstrate the existence of major morphological and physiological differences among populations of F. concolor , a finding potentially related to differences in genetic diversity.

Molecular evidence for species-level distinctions in clouded leopards.

Among the 37 living species of Felidae, the clouded leopard (Neofelis nebulosa) is generally classified as a monotypic genus basal to the Panthera lineage of great cats. This secretive, mid-sized (16-23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A). We used molecular genetic methods to re-evaluate subspecies partitions and to quantify patterns of population genetic variation among 109 clouded leopards of known geographic origin (Figure 1A, Tables S1 ans S2 in the Supplemental Data available online). We found strong phylogeographic monophyly and large genetic distances between N. n. nebulosa (mainland) and N. n. diardi (Borneo; n = 3 individuals) with mtDNA (771 bp), nuclear DNA (3100 bp), and 51 microsatellite loci. Thirty-six fixed mitochondrial and nuclear nucleotide differences and 20 microsatellite loci with nonoverlapping allele-size ranges distinguished N. n. nebulosa from N. n. diardi. Along with fixed subspecies-specific chromosomal differences, this degree of differentiation is equivalent to, or greater than, comparable measures among five recognized Panthera species (lion, tiger, leopard, jaguar, and snow leopard). These distinctions increase the urgency of clouded leopard conservation efforts, and if affirmed by morphological analysis and wider sampling of N. n. diardi in Borneo and Sumatra, would support reclassification of N. n. diardi as a new species (Neofelis diardi).

Approaches and efficacy of artificial insemination in felids and mustelids

Two of the most diverse and species-rich families in the order Carnivora (containing 280 species) include Felidae (cats) and Mustelidae (commonly called weasels). According to a current taxonomic classification system, the family Felidae consists of 41 felid species, most of which are listed as threatened or endangered with extinction [1]. The family Mustelidae consists of 59 species (including weasels, ferrets, minks, badgers, otters, wolverines), with the North American black-footed ferret (Mustela nigripes) being the most endangered [1]. Ex situ breeding programs exist for many of these rare carnivores, and one of the highest priorities is the retention of existing gene diversity to ensure species integrity, health and reproduction. Tools associated with ‘assisted reproduction’ offer multiple advantages for species propagated under the auspices of an organized genetic management plan (e.g., the Species Survival Plan or SSP#; Table 1) [2,3]. Because cooperating institutions (usually zoos) breed animals on the basis of the genetic value of individuals and computerized calculations of

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa☆

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors

Spermatozoa from teratospermic domestic cats (>60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (<40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95‐ and 160‐kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG‐50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187‐induced AR; (4) the spontaneous and zona pellucida (ZP)–induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP‐intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95‐ and 160‐kDa) sperm proteins. Although both inhibitors blocked the ZP‐induced AR, neither influenced the spontaneous AR nor the A23187‐induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor‐treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP‐induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase–dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males. Mol. Reprod. Dev. 49:48–57, 1998.

Age-Dependent Changes in Sperm Production, Semen Quality, and Testicular Volume in the Black-Footed Ferret (Mustela nigripes)

Abstract The black-footed ferret (Mustela nigripes), which was extirpated from its native North American prairie habitat during the 1980s, is being reintroduced to the wild because of a successful captive-breeding program. To enhance propagation, the reproductive biology of this endangered species is being studied intensively. The typical life span of the black-footed ferret is approximately 7 yr. Female fecundity declines after 3 yr of age, but the influence of age on male reproduction is unknown. In this study, testis volume, seminal traits, sperm morphology, and serum testosterone were compared in 116 males from 1 to 7 yr of age living in captivity. Results demonstrated that testes volume during the peak breeding season was similar (P > 0.05) among males 1 to 5 yr of age, reduced (P < 0.05) among males 6 yr of age, and further reduced (P < 0.05) among males 7 yr of age. Motile sperm/ejaculate was similar in males 1 to 6 yr of age but diminished (P < 0.05) in those 7 yr of age. Males at 6 and 7 yr of age produced fewer (P < 0.05) structurally normal sperm than younger counterparts; however, serum testosterone concentrations were not reduced (P > 0.05) in older males. Histological comparison of testicular/epididymal tissue from 5- and 7-yr-old black-footed ferrets confirmed that the interval between these two ages may represent a transitional period to reproductive senescence. In summary, functional reproductive capacity of male black-footed ferrets exceeds that of females by at least 2 yr. Testes and seminal quality are indistinguishable among males 1 to 5 yr of age, with progressive reproductive aging occurring thereafter.

Ejaculate traits in the Namibian cheetah (Acinonyx jubatus): influence of age, season and captivity

The objective was to examine the influence of animal age, season and captivity status on seminal quality in wild-born cheetahs (Acinonyx jubatus) in Namibia, Africa. Animals were divided into three age categories: juvenile (14-24 months; n = 16 males, 23 ejaculates); adult (25-120 months; n = 76 males, 172 ejaculates); and aged (>120 months; n = 5 males, 5 ejaculates). Seasons were categorised into hot-wet (January-April), cold-dry (May-August) and hot-dry (September-December). A comparison between freshly wild-caught (n = 29 males, 41 ejaculates) and captive-held cheetahs (n = 68 males, 159 ejaculates) was also conducted. Raw ejaculates contained 69.0 +/- 1.1% motile spermatozoa (mean +/- s.e.m.) with 73.6 +/- 1.5% of these cells containing an intact acrosome. Overall, 18.4 +/- 0.9% of spermatozoa were morphologically normal, with midpiece anomalies being the most prevalent (approximately 39%) defect. Juvenile cheetahs produced ejaculates with poorer sperm motility, forward progressive status, lower seminal volume and fewer total motile spermatozoa than adult and aged animals. Spermatogenesis continued unabated throughout the year and was minimally influenced by season. Proportions of sperm malformations were also not affected by season. Ejaculates from captive cheetahs had increased volume and intact acrosomes, but lower sperm density than wild-caught counterparts. In summary, Namibian cheetahs produce an extraordinarily high proportion of pleiomorphic spermatozoa regardless of age, season or living (captive versus free-ranging) status. Young males less than 2 years of age produce poorer ejaculate quality than adult and aged males. Because (1) all study animals were wild born and (2) there was little difference between freshly caught males and those maintained in captivity for protracted periods, our results affirm that teratospermia in the cheetah is mostly genetically derived. It also appears that an ex situ environment for the Namibian cheetah can ensure sperm quality comparable with that for free-living males.

Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant☆

The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat [normospermic versus teratospermic], the normospermic serval and the teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Hams F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (P<0.01) sperm motility compared to control spermatozoa maintained in Hams F10. Exposing sperm from all species to a 1M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Hams F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.