Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

Influence of breed on blood and tissue copper status in growing and finishing steers fed diets supplemented with copper.

To evaluate the influence of breed on the accumulation of dietary copper (Cu) in tissue, and on blood parameters indicative of Cu status, ten Galician Blond, nine Holstein-Friesian and ten Galician Blond x Holstein-Friesian cross (GB x HF) steers were fed diets supplemented with 35 mg/kg DM of CuSO4 during their growing and finishing periods. Blood samples were taken monthly, and samples of liver, kidney, brain, heart, spleen and muscle were taken at slaughter. Cu concentrations were determined by ICP-AES. Holstein-Friesian calves had significantly higher total liver Cu contents than Galician Blonds and GB x HF crosses (mean 1070, 663 and 868 mg, respectively), combined with higher hepatic Cu concentrations (174, 140 and 166 mg/kg wet weight, respectively). Holstein-Friesian calves had also the highest prevalence (89%) of hepatic Cu concentrations exceeding the toxic limit of 150 mg/kg wet weight. Breed did not have a statistically significant influence on blood parameters. With the exception of the semitendinosus muscle, where Holstein-Friesians (0.790 mg/kg) had significantly higher Cu levels than Galician Blonds (0.541 mg/kg) or GB x HF crosses (0.631 mg/kg), no other statistically significant differences by breed in the extrahepatic tissue Cu distribution were observed. A negative statistical association between carcass performance and the ratio of semitendinosus and liver Cu concentration could indicate that the animals with a better carcass performance (Galician Blonds) could need a higher Cu mobilisation into the muscle, resulting in a lower hepatic storage.

The interlobular distribution of copper in the liver of beef calves on a high-copper diet.

The aims of the present study were 1) to evaluate the interlobular distribution of copper (Cu) in the liver of beef calves on a high-Cu diet, 2) to determine whether this distribution differs between Galician Blonds and Holstein Friesians, and 3) to determine whether in vivo needle biopsy provides an appropriate measure of overall hepatic Cu status. Liver biopsies were performed before slaughter on twenty-nine 10-month-old beef calves fed growing and finishing diets supplemented with 35 mg/kg of Cu sulfate (10 Galician Blonds, 9 Holstein Friesians, and 10 Galician Blond x Holstein Friesian crosses). At slaughter, samples taken from 6 regions of the liver (the internal and external faces of the right lobe; the left, caudate, and quadrate lobes; and the processus papillaris) were acid digested, and their Cu contents were determined by inductively coupled plasma atomic emission spectrometry. The highest Cu concentrations were found in the left lobe, followed by the processus papillaris, and the lowest Cu concentrations were found in the caudate and quadrate lobes. Different breeds differ in absolute hepatic Cu levels, but interlobular Cu distribution does not appear to depend on breed, at least when Galician Blonds are compared with Holstein Friesians. In vivo needle biopsy afforded accurate estimates of overall hepatic Cu status.