The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.

Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation.

The horse has been a food source, but more importantly, it has been a means for transport. Its domestication was one of the crucial steps in the history of human civilization. Despite the archaeological and molecular studies carried out on the history of horse domestication, which would contribute to conservation of the breeds, the details of the domestication of horses still remain to be resolved. We employed 21 microsatellite loci and mitochondrial control region partial sequences to analyse genetic variability within and among four Anatolian native horse breeds, Ayvacık Pony, Malakan Horse, Hınıs Horse and Canik Horse, as well as samples from indigenous horses of unknown breed ancestry. The aims of the study were twofold: first, to produce data from the prehistorically and historically important land bridge, Anatolia, in order to assess its role in horse domestication and second, to analyse the data from a conservation perspective to help the ministry improve conservation and management strategies regarding native horse breeds. Even though the microsatellite data revealed a high allelic diversity, 98% of the genetic variation partitioned within groups. Genetic structure did not correlate with a breed or geographic origin. High diversity was also detected in mtDNA control region sequence analysis. Frequencies of two haplogroups (HC and HF) revealed a cline between Asia and Europe, suggesting Anatolia as a probable connection route between the two continents. This first detailed genetic study on Anatolian horse breeds revealed high diversity among horse mtDNA haplogroups in Anatolia and suggested Anatolia's role as a conduit between the two continents. The study also provides an important basis for conservation practices in Turkey.