Global status of gene edited animals for agricultural applications.

Gene editing (GnEd) involves using a site-directed nuclease to introduce a double-strand break (DSB) at a targeted location in the genome. A literature search was performed on the use of GnEd in animals for agricultural applications. Data was extracted from 212 peer-reviewed articles that described the production of at least one living animal employing GnEd technologies for agricultural purposes. The most common GnEd system reported was CRISPR/Cas9, and the most frequent type of edit was the unguided insertion or deletion resulting from the repair of the targeted DSB leading to a knock-out (KO) mutation. Animal groups included in the reviewed papers were ruminants (cattle, sheep, goats, n=63); monogastrics (pigs and rabbits, n=60); avian (chicken, duck, quail, n=17); aquatic (many species, n=65), and insects (honeybee, silkworm, n=7). Yield (32%), followed by reproduction (21%) and disease resistance (17%) were the most commonly targeted traits. Over half of the reviewed papers had Chinese first-authorship. Several countries, including Argentina, Australia, Brazil, Colombia and Japan, have adopted a regulatory policy that considers KO mutations introduced following GnEd DSB repair as akin to natural genetic variation, and therefore treat these GnEd animals analogously to those produced using conventional breeding. This approach has resulted in a non-GMO determination for a small number of GnEd food animal applications, including three species of GnEd KO fast-growing fish, (red sea bream, olive flounder and tiger pufferfish in Japan), KO fish and cattle in Argentina and Brazil, and porcine reproductive and respiratory syndrome (PRRS) virus disease-resistant KO pigs in Colombia.

Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes.

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

Review: Progress in producing chimeric ungulate livestock for agricultural applications.

The progress made in recent years in the derivation and culture of pluripotent stem cells from farm animals opens up the possibility of creating livestock chimeras. Chimeras producing gametes exclusively derived from elite donor stem cells could pass superior genetics on to the next generation and thereby reduce the genetic lag that typically exists between the elite breeding sector and the commercial production sector, especially for industries like beef and sheep where genetics is commonly disseminated through natural service mating. Chimeras carrying germ cells generated from genome-edited or genetically engineered pluripotent stem cells could further disseminate useful genomic alterations such as climate adaptation, animal welfare improvements, the repair of deleterious genetic conditions, and/or the elimination of undesired traits such as disease susceptibility to the next generation. Despite the successful production of chimeras with germ cells generated from pluripotent donor stem cells injected into preimplantation-stage blastocysts in model species, there are no documented cases of this occurring in livestock. Here, we review the literature on the derivation of pluripotent stem cells from ungulates, and progress in the production of chimeric ungulate livestock for agricultural applications, drawing on insights from studies done in model species, and discuss future possibilities of this fast-moving and developing field. Aside from the technical aspects, the consistency of the regulatory approach taken by different jurisdictions towards chimeric ungulate livestock with germ cells generated from pluripotent stem cells and their progeny will be an important determinant of breeding industry uptake and adoption in animal agriculture.

Recombinant TrxAFNIIx4His6 improves post-thaw motility of ram sperm measured by a sperm motility tracker software.

The aim of the present study was to evaluate a freezing extender supplemented with recombinant TrxAFNIIx4His6, a reported decapacitating factor. Semen samples were diluted in tris-egg yolk medium with 0, 1.5 µM and 3.0 µM of TrxAFNIIx4His6. Computer-assisted sperm motility tracking and subpopulations evaluation showed that addition of TrxAFNIIx4His6 improved post-thaw total and progressive motility at both concentrations evaluated. TrxAFNIIx4His6 increased the sperm subpopulation with the highest progressiveness and great velocity and decreased the subpopulation of poorly motile and almost non-progressive sperm. Incorporation of TrxAFNIIx4His6 to freezing extender shows potential for the development of cryoprotection media which may lead to improved fertility after artificial insemination.

Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization.

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ±â€¯2.95; 40.13 ±â€¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables.

Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters.