Effects of Cryoprotectant Concentration and Exposure Time during Vitrification of Immature Pre-Pubertal Lamb Cumulus-Oocyte Complexes on Nuclear and Cytoplasmic Maturation.

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Heterogeneity of a dwarf phenotype in Dutch traditional chicken breeds revealed by genomic analyses.

The growth of animals is a complex trait, in chicken resulting in a diverse variety of forms, caused by a heterogeneous genetic basis. Bantam chicken, known as an exquisite form of dwarfism, has been used for crossbreeding to create corresponding dwarf counterparts for native fowls in the Dutch populations. Here, we demonstrate the heterogeneity of the bantam trait in Dutch chickens and reveal the underlying genetic causes, using whole-genome sequence data from matching pairs of bantam and normal-sized breeds. During the bantam-oriented crossbreeding, various bantam origins were used to introduce the bantam phenotype, and three major bantam sources were identified and clustered. The genome-wide association studies revealed multiple genetic variants and genes associated with bantam phenotype, including HMGA2 and PRDM16, genes involved in body growth and stature. The comparison of associated variants among studies illustrated differences related to divergent bantam origins, suggesting a clear heterogeneity among bantam breeds. We show that in neo-bantam breeds, the bantam-related regions underwent a strong haplotype introgression from the bantam source, outcompeting haplotypes from the normal-sized counterpart. The bantam heterogeneity is further confirmed by the presence of multiple haplotypes comprising associated alleles, which suggests the selection of the bantam phenotype is likely subject to a convergent direction across populations. Our study demonstrates that the diverse history of human-mediated crossbreeding has contributed to the complexity and heterogeneity of the bantam phenotype.

Ex situ and in situ data for endangered livestock breeds in Spain.

Improvements in ex situ storage of genetic and reproductive materials offer an alternative for endangered livestock breed conservation. This paper presents a dataset for current ex situ collections and in situ population for 179 Spanish livestock breeds of seven species, cattle, sheep, pig, chicken, goat, horse and donkey. Ex situ data was obtained via survey administered to 18 functioning gene banks in Spain and relates to the reproductive genetic materials (semen doses) of 210 livestock breeds distributed across the gene banks. In situ data combines CENSUS information with linear regression techniques and relates to the geographic distribution of 179 Spanish autochthonous livestock breeds (2009-2018), and in situ population projections and extinction probabilities (2019-2060). We use a decision variable defining an "acceptable level of risk" that allows decision makers to specify tolerable levels of in situ breed endangerment when taking ex situ collection and storage decisions.

Quality Management Practices of Gene Banks for Livestock: A Global Review.

The genetic diversity of livestock is decreasing and many countries have created gene banks for ex situ-in vitro conservation of animal genetic resources (AnGR). The collection, processing, and storage of animal germplasm require substantial investment and the material collected (and associated data) is highly valuable. Therefore, quality management systems (QMSs) and practices are important. The objective of this study was to review the quality management procedures of livestock gene banks around the world to identify the general strengths and weaknesses of quality control. A survey was administered by means of an online questionnaire consisting of 54 questions, most of which were yes/no with respect to the presence of a particular aspect of quality management. The survey was distributed through networks of the Food and Agriculture Organization of the United Nations that are associated with AnGR. Ninety responses were received from 62 countries. The gene banks were predominantly public institutions, with the main goal of preventing breed extinction. Approximately 30% of the banks reported having a QMS, 15 of which involved formal certification. Many other banks have plans to implement formal quality management within the next 5 years. Regarding specific aspects of quality management, more emphasis was placed on material entering the banks than on eventual utilization. Among the banks processing and freezing material, 90% followed specific standard operating procedures, but only 24% had policies regarding provision of access to external stakeholders. Increased cooperation among livestock gene banks could improve quality management. Sharing of knowledge could standardize procedures and cooperating peers could evaluate each other's QMSs.

Optimizing ex situ genetic resource collections for European livestock conservation.

Ex situ collections offer the potential to reduce extinction risks, affording option to society in maintaining future breeding opportunities for productivity and heritage traits. However, how much should we be seeking to collect and conserve in gene banks, and where? We developed a mathematical model to optimize logistical decisions of breed conservation choices and to evaluate alternative scenarios for efficiently re-allocating genetic materials currently stored in different European gene banks, allowing for cross-country collections, cost and cryogenic capacity differentials. We show how alternative allocations for the breeds that are currently stored in 11 European gene banks could reduce overall conservation costs by around 20% by selecting cryogenic banks that have relatively lower combination of fixed and collection costs, and are geographically closer to collection regions. Our results show that centralizing collections in one gene bank would double the costs, relative to collective European collections approaches. We also calculate marginal costs of collections and show that increasing diversity within the gene banks implies in higher costs per conserved breed.

The impact of using old germplasm on genetic merit and diversity-A cattle breed case study.

Artificial selection and high genetic gains in livestock breeds led to a loss of genetic diversity. Current genetic diversity conservation actions focus on long-term maintenance of breeds under selection. Gene banks play a role in such actions by storing genetic materials for future use and the recent development of genomic information is facilitating characterization of gene bank material for better use. Using the Meuse-Rhine-Issel Dutch cattle breed as a case study, we inferred the potential role of germplasm of old individuals for genetic diversity conservation of the current population. First, we described the evolution of genetic merit and diversity over time and then we applied the optimal contribution (OC) strategy to select individuals for maximizing genetic diversity, or maximizing genetic merit while constraining loss of genetic diversity. In the past decades, genetic merit increased while genetic diversity decreased. Genetic merit and diversity were both higher in an OC scenario restricting the rate of inbreeding when old individuals were considered for selection, compared to considering only animals from the current population. Thus, our study shows that gene bank material, in the form of old individuals, has the potential to support long-term maintenance and selection of breeds.

The effects of recent changes in breeding preferences on maintaining traditional Dutch chicken genomic diversity.

Traditional Dutch chicken breeds are marginalised breeds of ornamental and cultural-historical importance. In the last decades, miniaturising of existing breeds (so called neo-bantam) has become popular and resulted in alternatives to original large breeds. However, while backcrossing is increasing the neo-bantams homozygosity, genetic exchange between breeders may increase their genetic diversity. We use the 60 K SNP array to characterise the genetic diversity, demographic history, and level of inbreeding of Dutch heritage breeds, and particularly of neo-bantams. Commercial white layers are used to contrast the impact of management strategy on genetic diversity and demography. A high proportion of alleles was found to be shared between large fowls and neo-bantams, suggesting gene flow during neo-bantams development. Population admixture analysis supports these findings, in addition to revealing introgression from neo-bantams of the same breed and of phenotypically similar breeds. The prevalence of long runs of homozygosity (ROH) confirms the importance of recent inbreeding. A high diversity in management, carried out in small breeding units explains the high heterogeneity in diversity and ROH profile displayed by traditional breeds compared to commercial lines. Population bottlenecks may explain the long ROHs in large fowls, while repetitive backcrossing for phenotype selection may account for them in neo-bantams. Our results highlight the importance of using markers to inform breeding programmes on potentially harmful homozygosity to prevent loss of genetic diversity. We conclude that bantamisation has generated unique and identifiable genetic diversity. However, this diversity can only be preserved in the near future through structured breeding programmes.

Cryopreservation of turkey semen: effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching.

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2×2×2×5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8-31.5%) of fertile, non-viable embryos (Day 1-6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10-13 weeks and 9-10 weeks, respectively. The duration of hatchability was 4-6 weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5±2.4%), followed by the E line (5.3±1.3%), F line (3.7±2.0%) and RBC2 line (2.6±0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.