Search

IntroductionFreeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage.MethodsThis work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C.Results and discussionThe results demonstrated better structural stability, namely lipid composition and DNA integrity, in VDE spermatozoa than FD ones, with outcomes at RT storage comparable to 4°C. Likewise, in VDE the embryonic development was higher than in FD samples (12.8% vs. 8.7%, p < 0.001, respectively). Our findings indicated that in large mammals, it is important to consider dehydration-related changes in sperm polyunsaturated fatty acids coupled with DNA alterations, given their crucial role in embryonic development.

Abstract The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P

Local breeds represent an underestimated resource in terms not only of their important cultural and economical role in marginal areas, but also because they often own a potential genetic pool well adapted to extreme conditions. This fact is of increasing interest, especially when considering climate global challenges where peculiar and uncommon traits could be advantageous. In this study, we genotyped 24 individuals belonging to the small residual Pagliarola sheep population using the OvineSNP50K array, in order to compare its genomic architecture with other 21 Italian local breeds. Moreover, we performed the fixation index (FST) outlier analysis to identify genes most differentiated between Pagliarola and Merino-derived Italian breeds. All population genetic analyses highlighted that Pagliarola breed represents a distinct genetic unit showing a genetic relationship with Merino-derived breeds underlying the close cultural connection between these breeds related to transhumance. However, Pagliarola breed resulted to be divided into two different sub-populations, named Pagliarola 1 (PAG-1) and Pagliarola 2 (PAG-2). Genetic diversity indices and inbreeding estimated from runs of homozygosity (FROH) indicated that while PAG-1 showed among the highest values, PAG-2 turned out strongly inbred, probably as a consequence of a founder effect. The FST outlier analysis identified the 5 most differentiated single nucleotide polymorphisms, two of which mapping within known genes (MACF1 and GLIS1) reported to be linked to feed efficiency and local adaption. All these information strongly suggest that proper conservation measures should be implemented in order to recover the Pagliarola population and its fundamental local products.HIGHLIGHTS All population genetic analyses highlighted that Pagliarola breed represent a distinct genetic unit, and clusters with the Merino-derived Italian breeds. The breed resulted to be divided into two different sub-populations. For the two Pagliarola sub-populations, the genetic diversity indices suggest a very different genetic makeup. Results from the Bayesian approach identified two know genes (MACF1and GLIS1) related to feed efficiency in livestock species and local adaptation. The information generated in this study strongly suggests that proper conservation measures should be implemented in order to recover the Pagliarola population.