L. A. Johnston

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.

Influence of culture medium and protein supplementation on in vitro oocyte maturation and fertilization in the domestic cat

Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagles Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17beta and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.

Molecular Genetic Characterization of Two Insular Asian Cat Species, Bornean Bay Cat and Iriomote Cat

Molecular genetic data were used to characterize the genetic distinctiveness of Borncan bay cat (Pardofelis badia) and Iriomote cat (Prionailurus bengalensis iriomotensis), small cat species restricted to separate Asian islands. Sequence variation in two mitochondrial genes, NADH dehydrogenase subunit 5 (NADH-5) and ATPase-8 (ATP-8) was used to examine the phylogenetic relationship between a recently discovered Bornean bay cat specimen and the original type specimen (collected in 1855) relative to other Southeast Asian felids. DNA and amino acid sequence analyses affirmed that both bay cat specimens derived from the same phylogenetic lineage and that Bornean bay cat shared a monophyletic common ancestor with Asian golden cat (Profelis temmincki) estimated at 4.9-5.3 million years ago, well before the geological separation of Borneo from mainland Asia which occurred in the late Pleistocene, estimated as 10,000-20,000 years ago. The phylogenetic distinctiveness of the Iriomote cat (Prionailurus iriomotensis or P. bengalensis iriomotensis, n=5) from two leopard cat subspecies (P. b. euptilurus, n=5 and P. b. bengalensis, n=13) was examined based upon the DNA sequence variation of four mitochondrial genes, NADH-5, ATP-8, 16S rRNA, and Cytochrome b and based upon allele variation at 18 nuclear microsatellite loci. The available sample of Iriomote cats displayed a remarkable reduction in overall genetic diversity from diversity in both mtDNA and microsatellite variation compared to other felids. Nonetheless, the Iriomote cat genes clearly aligned them with, but distinct from, other subspecies of leopard cat (P. b. euptilurus and P. b. bengalensis) affirming their taxonomic classification as P. b. iriomotensis, subspecies. The contrasting patterns of the genetic variation of Bornean bay cat and Iriomote cat likely reflect different natural histories for these two island cat taxa.