56 EFFECTS OF PHYLOGENIC GENERA OF RECIPIENT CYTOPLASTS ON DEVELOPMENT AND VIABILITY OF CANADA LYNX (LYNX CANADENSIS) CLONED EMBRYOS

Abstract

Canada lynx (Lynx canadensis; CL) once occupied 16 states in the Unites States of America, but small populations remain in only 3 states. Interspecies-somatic cell nuclear transfer (Is-SCNT) offers the possibility of preventing their extinction; however, developmental constraints on Is-SCNT embryos are proportional to the phylogenetic distance between the donor cell and the recipient oocyte. Mitochondrial DNA (mtDNA) heteroplasmy may be involved in nuclear-cytoplasmic incompatibilities, thus inhibiting development of cloned embryos at the time of genomic activation. Minimizing the phylogenetic distance between the donor cell and recipient oocyte may enhance development of clone embryos. Caracal (Caracal caracal) may be suitable as an oocyte donor for SCNT and a recipient of CL cloned embryos because caracals hybridize with other felid species and share physical characteristics with the lynx family, marked by being previously classified in the lynx genera and having similar gestational length. To ensure compatibilities between the donor nuclei of the CL and the mitochondria of recipient oocytes, we (1) compared in vitro development of CL cloned embryos reconstructed with domestic cat (Felis catus; DSH) or caracal cytoplasts, (2) examined the mtDNA genotypes in CL cloned embryos, and (3) evaluated in vivo developmental competence of CL cloned embryos after transfer into caracal recipients. A total of 160 and 217 preovulatory oocytes were collected by laparoscopy from gonadotropin-treated caracals (n = 8) and DSH (n = 10) and used as recipient cytoplasts for reconstructing CL embryos. Results indicated that the phylogenetic genera of recipient cytoplasts did not affect embryo cleavage at Day 2 (caracal 50/55, 91% v. DSH 63/65, 97%), but development of CL cloned embryos to the blastocyst stage was higher when caracal oocytes were used as recipient cytoplasts (15/50; 30%) than with DSH cytoplasts (9/63, 14%; P < 0.05). The extent of mtDNA homoplasmy or heteroplasmy in CL cloned embryos was calculated by the number of single nucleotide polymorphisms (SNP) derived from the DSH or caracal oocyte donors and from the somatic cell donor CL. DNA was isolated from 25 and 35 CL cloned embryos reconstructed with caracal or DSH cytoplasts, respectively. All amplified products after PCR were sequenced and SNP analyzed. All CL embryos reconstructed with DSH cytoplasts were homoplasmic, carrying mtDNA only from the DSH oocyte donor (n = 35; SNP DSH = 2-6). Embryos reconstructed with caracal cytoplasts were homoplasmic for CL mtDNA (n = 9; SNPCL = 10-12) or heteroplasmic (caracal × CL, n = 17; SNPCL = 7-9; SNP caracal = 2-3). A total of 69 (mean = 34.5 ± 4.9 per caracal) and 70 (mean = 35.0 ± 9.8 per caracal) CL cloned embryos reconstructed with caracal and DSH cytoplasts, respectively, were transferred into 4 caracal recipients; however, no pregnancies were established. In summary, Is-SCNT between 2 phylogenetically closer species favors retention of the donor’s mitochondria, which might lead to a better nucleo-cytoplasmic interaction for reprogramming of donor nucleus.