In Vitro Function of Frozen-Thawed Bottlenose Dolphin (Tursiops truncatus) Spermatozoa Undergoing Sorting and Recyopreservation

Artificial insemination (AI) with sex-sorted bottlenose dolphin spermatozoa provides female calves for obtaining more cohesive social groups and optimum genetic management of captive populations. However, distance of animals to the sorting facility represents a limit to the procedure. Although one bottlenose dolphin calf has been born using spermatozoa from frozen-thawed, sorted and recryopreserved spermatozoa, critical evaluation of the steps involved in this process is required to maximize its efficiency for future AIs and expansion of the technology to other species. Two experiments were designed to determine the efficiency of the sorting process and the quality of frozen-thawed bottlenose dolphin spermatozoa during sorting and recryopreservation. In experiment 1, the effect of two washing media (with and without 4 percent egg yolk, v/v) following density gradient centrifugation (DGC) on sperm recovery rate and in vitro characteristics of cryopreserved spermatozoa was examined. In experiment 2, cryopreserved semen was used to compare the effects of two recryopreservation methods (conventional straw freezing and directional freezing) on in vitro sperm characteristics of control (non-sorted) and sorted spermatozoa. Egg yolk supplementation of the washing medium in experiment 1 did not influence (P > 0.05) the sperm recovery rate, however, sperm motility parameters and viability were improved (P < 0.05). For Experiment 2, motility parameters and viability were influenced by stage of sex-sorting process, sperm type (non-sorted and sorted) and freezing method (P < 0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P < 0.05) motility parameters over the 24 h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of nonsorted spermatozoa, as assessed by the SCSA, remained unchanged throughout the process. However, a possible interaction between Hoechst 33342 and acridine orange was observed in sorted samples. After recryopreservation, viability of sorted spermatozoa was higher (P < 0.05) than that of non-sorted spermatozoa across all time points. The percentages of viable spermatozoa determined by light (eosin-nigrosin) and fluorescence microscopy (propidium iodide) techniques were correlated (R^2=0.79, P < 0.001). Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI

Partial cDNA sequence analysis of myosin Va from rainbow trout (Oncorhynchus mykiss) and its relationship to myosin v isoforms from other vertebrates.

Partial cDNA sequences of myosin V from rainbow trout Oncorhynchus mykiss were analyzed and showed high similarity to MVa from other vertebrates. Phylogenetic analysis has shown that events resulting in the formation of paralogous copies of myosin Va, Vb, and Vc occurred before the divergence of vertebrates into different classes. Expression analysis of myosin Va, Vb, and Vc in different O. mykiss tissues revealed MVa exclusively expressed in hypophysis and brain whereas Vb and Vc were expressed in practically all tissues analyzed. The nucleotide sequence for myosin V was explored in a fish species for the first time and these results represent an important start in understanding the organization, evolution, and expression of myosins in early vertebrates. The data presented here represent contributions to the knowledge of rainbow trout genome. A better understanding of this economically important species could assist in development of improved strains of this fish for aquaculture

Brain distribution of myosin Va in rainbow trout Oncorhynchus mykiss

This study presents data on myosin Va localization in the central nervous system of rainbow trout. We demonstrate, via immunoblots and immunocytochemistry, the expression of myosin Va in several neuronal populations of forebrain, midbrain, hindbrain and spinal cord. The neuronal populations that express myosin Va in trout constitute a very diverse group that do not seem to have many specific similarities such as neurotransmitters used, cellular size or length of their processes. The intensity of the immunoreactivity and the number of immunoreactive cells differ from region to region. Although there is a broad distribution of myosin Va, it is not present in all neuronal populations. This result is in agreement with a previous report, which indicated that myosin Va is approximately as abundant as conventional myosin II and kinesin, and it is broadly involved in neuronal motility events such as axoplasmatic transport. Furthermore, this distribution pattern is in accordance with what was shown in rats and mice; it indicates phylogenetic maintenance of the myosin Va main functions

Multi-tissue DNA methylation aging clocks for sea lions, walruses and seals.

Age determination of wild animals, including pinnipeds, is critical for accurate population assessment and management. For most pinnipeds, current age estimation methodologies utilize tooth or bone sectioning which makes antemortem estimations problematic. We leveraged recent advances in the development of epigenetic age estimators (epigenetic clocks) to develop highly accurate pinniped epigenetic clocks. For clock development, we applied the mammalian methylation array to profile 37,492 cytosine-guanine sites (CpGs) across highly conserved stretches of DNA in blood and skin samples (n = 171) from primarily three pinniped species representing the three phylogenetic families: Otariidae, Phocidae and Odobenidae. We built an elastic net model with Leave-One-Out-Cross Validation (LOOCV) and one with a Leave-One-Species-Out-Cross-Validation (LOSOCV). After identifying the top 30 CpGs, the LOOCV produced a highly correlated (r = 0.95) and accurate (median absolute error = 1.7 years) age estimation clock. The LOSOCV elastic net results indicated that blood and skin clock (r = 0.84) and blood (r = 0.88) pinniped clocks could predict age of animals from pinniped species not used for clock development to within 3.6 and 4.4 years, respectively. These epigenetic clocks provide an improved and relatively non-invasive tool to determine age in skin or blood samples from all pinniped species

DNA methylation networks underlying mammalian traits

Using DNA methylation profiles ( n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species

DNA methylation networks underlying mammalian traits

Using DNA methylation profiles ( = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species