Growth analysis and blood profile in piglets born by embryo transfer

©2022. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Published, version of a Published Work that appeared in final form in Research in Veterinary Science. To access the final edited and published work see https://doi.org/10.1016/j.rvsc.2021.11.010Assisted reproductive technologies (ART), besides solving several reproductive problems, it has also been used as a tool to improve the animal productivity that is required for feeding the human population. One of these techniques, the embryo transfer (ET), has presented limitations in the porcine species, which could constrain its use in the porcine industry. To clarify the potential of this technique, we aimed to compare the impact of using ET or artificial insemination (AI) on the phenotype of the offspring during its first days of age, in terms of growth and blood parameters. At birth, the body weight was higher for ET-females than AI-females, but this difference was no longer observed at day 15. On day 3, it was observed a higher concentration of red blood cells, haemoglobin, and haematocrit in females-ET and a higher concentration of white blood cells in both ET-derived piglets (males and females) when compared to AI groups. On day 3, the biochemical analysis showed a higher level of albumin for ET-derived males, and a lower level of bilirubin for ET-females than AI controls. However, all values were within the normal ranges. Our results indicate that piglets derived from ET seem to be phenotypically similar to those born by AI, which provides preliminary evidence that the ET procedure is a safe technique, but additional studies beyond 15 days of life are requested to conclude its global impact. Furthermore, the presented reference values of blood parameters in this species are interesting data for the pig industry

Growth analysis and blood profile in piglets born by embryo transfer

Assisted reproductive technologies (ART), besides solving several reproductive problems, it has also been used as a tool to improve the animal productivity that is required for feeding the human population. One of these techniques, the embryo transfer (ET), has presented limitations in the porcine species, which could constrain its use in the porcine industry. To clarify the potential of this technique, we aimed to compare the impact of using ET or artificial insemination (AI) on the phenotype of the offspring during its first days of age, in terms of growth and blood parameters. At birth, the body weight was higher for ET-females than AI-females, but this difference was no longer observed at day 15. On day 3, it was observed a higher concentration of red blood cells, haemoglobin, and haematocrit in females-ET and a higher concentration of white blood cells in both ET-derived piglets (males and females) when compared to AI groups. On day 3, the biochemical analysis showed a higher level of albumin for ET-derived males, and a lower level of bilirubin for ET-females than AI controls. However, all values were within the normal ranges. Our results indicate that piglets derived from ET seem to be phenotypically similar to those born by AI, which provides preliminary evidence that the ET procedure is a safe technique, but additional studies beyond 15 days of life are requested to conclude its global impact. Furthermore, the presented reference values of blood parameters in this species are interesting data for the pig industry

Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM

Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits. Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.MINECO, FEDER, Fundación Senec

Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation

Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination

Abstract Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2’s pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected be- fore insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.MINECO-FEDER, Fundación Senec

One-Step In Vitro Generation of ETV2-Null Pig Embryos

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs