Sperm cryopreservation and artificial insemination are important methods for giant panda breeding and preservation of extant genetic diversity. Lower conception rates limit the use of artificial insemination with frozen-thawed giant panda sperm, due to the lack of understanding of the cryodamaging or cryoinjuring mechanisms in cryopreservation. Long non-coding RNAs (lncRNAs) are involved in regulating spermatogenesis. However, their roles during cryopreservation remain largely unexplored. Therefore, this study aimed to identify differentially expressed lncRNAs and mRNAs associated with cryodamage or freeze tolerance in frozen-thawed sperm through high throughput sequencing. A total of 61.05 Gb clean reads and 22,774 lncRNA transcripts were obtained. From the sequencing results, 1477 significantly up-regulated and 1,396 significantly down-regulated lncRNA transcripts from fresh and frozen-thawed sperm of giant panda were identified. GO and KEGG showed that the significantly dysregulated lncRNAs and mRNAs were mainly involved in regulating responses to cold stress and apoptosis, such as the integral component of membrane, calcium transport, and various signaling pathways including PI3K-Akt, p53 and cAMP. Our work is the first systematic profiling of lncRNA and mRNA in fresh and frozen-thawed giant panda sperm, and provides valuableinsights into the potential mechanism of cryodamage in sperm.

Post-thawed sperm quality parameters vary across different species after cryopreservation. To date, the molecular mechanism of sperm cryoinjury, freeze-tolerance and other influential factors are largely unknown. In this study, significantly dysregulated microRNAs (miRNAs) and mRNAs in boar and giant panda sperm with different cryo-resistance capacity were evaluated. From the result of miRNA profile of fresh and frozen-thawed giant panda sperm, a total of 899 mature, novel miRNAs were identified, and 284 miRNAs were found to be significantly dysregulated (195 up-regulated and 89 down-regulated). Combined analysis of miRNA profiling of giant panda sperm and our previously published data on boar sperm, 46, 21 and 4 differentially expressed (DE) mRNAs in boar sperm were believed to be related to apoptosis, glycolysis and oxidative phosphorylation, respectively. Meanwhile, 87, 17 and 7 DE mRNAs in giant panda were associated with apoptosis, glycolysis and oxidative phosphorylation, respectively. Gene ontology (GO) analysis of the targets of DE miRNAs showed that they were mainly distributed on membrane related pathway in giant panda sperm, while cell components and cell processes were tied to the targets of DE miRNAs in boar sperm. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DE mRNAs indicated that most of these DE mRNAs were distributed in membrane signal transduction-related pathways in giant panda sperm, while those in boar sperm were mainly distributed in the cytokine-cytokine receptor interaction pathway and inflammatory related pathways. In conclusion, although the different freezing extenders and programs were used, the DE miRNAs and mRNAs involved in apoptosis, energy metabolism, olfactory transduction pathway, inflammatory response and cytokine-cytokine interactions, could be the possible molecular mechanism of sperm cryoinjury and freeze tolerance.

Captive populations provide a precious genetic resource for endangered animals and a source of individuals for reintroduction to depleted habitats. Therefore, accuracy in determining paternity is of vital importance for managing captive populations and in selecting representative individuals of known genetic characteristics for release. In this study, we established a fast and effective method to conduct paternity testing for captive giant pandas in the Wolong population. This technique uses two highly polymorphic microsatellites initially, subsequent use of five less polymorphic markers and then paternity exclusion testing carried out using the giant panda paternity exclusion program we have developed. Our results revealed that (1) both sets of markers successfully identified the real fathers in 25 cases of paternity testing and (2) the success rate of paternity exclusion varied with the degree of polymorphism of the markers used. Subsequently, we conducted correlation analysis between the success rates of paternity identification with these markers, parameters of genetic diversity and tests of neutrality. We found that the paternity exclusion power of microsatellites was significantly correlated with the number of alleles (Na), expected heterozygosity (H _E) and observed homozygosity statistic (F _O) (all P < 0.05). From this, we developed a new variable, Na×H _E/F _O, showing a highly significant positive correlation with the resolution power of microsatellites (P = 0.001). Moreover, the first two highly polymorphic loci gave a 100% success rate of excluding non-paternal males because they yielded higher values of Na×H _E/F _O than the other five less polymorphic markers. Thus, the Na×H _E/F _O parameter appears suitable to serve as a criterion for selecting microsatellite markers, which could be used for high-resolution molecular techniques of paternity determination among a range of captive animals besides giant pandas.