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.

Reprint of: Gamma radiation effect on Trichinella pseudospiralis and Trichinella spiralis infected wild boar meat.

Trichinellosis is a foodborne parasitic disease caused by Trichinella spp. Different methods, such as cooking, freezing and irradiation, have been suggested to inactivate the parasite in meat infected with Trichinella spp. The International Commission on Trichinellosis (ICT) recommends an irradiation dose of 0.3 kGy to inactivate Trichinella spp. larvae, but its effectiveness in all Trichinella species has not yet been demonstrated. Therefore, the objective of the present study was to determine the effectiveness of gamma radiation in treating wild boar meat to inactivate Trichinella pseudospiralis and Trichinella spiralis. Two wild boars (Sus scrofa) were each inoculated per os (PO) with 20,000 muscle larvae (ML) of Trichinella. One wild boar was inoculated with T. pseudospiralis and the other one with T. spiralis. Both wild boars were euthanized 20 weeks post infection (pi). A 250░g sample from each selected muscle type (side ribs, shoulder and upper forelegs) was obtained in duplicate from each animal. One group of samples was not irradiated (Gc; n░=░6) while the other group of samples was irradiated (Gt; n░=░6) at the Atomic Centre of the Argentinean National Commission of Atomic Energy (CNEA) with a minimum and maximum dose of 0.32-0.41 kGy. Twenty gram of each muscle from Gt and Gc were taken at 24░h, 7 days, 14 days and 21 days post-irradiation, and all samples were individually processed by means of artificial digestion. The ML were then inoculated into mice to evaluate the intestinal phase and the muscular phase. All recovered larvae from Gt and Gc samples showed integrity of the cuticle and active motility. Adult worms and ML were recovered from all mice inoculated with ML from Gc. However, no adult worms or ML were obtained in mice inoculated with ML from Gt. These results show the efficacy of irradiation to inhibit the development of T. pseudospiralis and T. spiralis in the host, which could possibly be extended to other non-encapsulated species, but further studies are needed to demonstrate this hypothesis.

Gamma radiation effect on Trichinella pseudospiralis and Trichinella spiralis infected wild boar meat.

Trichinellosis is a foodborne parasitic disease caused by Trichinella spp. Different methods, such as cooking, freezing and irradiation, have been suggested to inactivate the parasite in meat infected with Trichinella spp. The International Commission on Trichinellosis (ICT) recommends an irradiation dose of 0.3 kGy to inactivate Trichinella spp. larvae, but its effectiveness in all Trichinella species has not yet been demonstrated. Therefore, the objective of the present study was to determine the effectiveness of gamma radiation in treating wild boar meat to inactivate Trichinella pseudospiralis and Trichinella spiralis. Two wild boars (Sus scrofa) were each inoculated per os (PO) with 20,000 muscle larvae (ML) of Trichinella. One wild boar was inoculated with T. pseudospiralis and the other one with T. spiralis. Both wild boars were euthanized 20 weeks post infection (pi). A 250 g sample from each selected muscle type (side ribs, shoulder and upper forelegs) was obtained in duplicate from each animal. One group of samples was not irradiated (Gc; n = 6) while the other group of samples was irradiated (Gt; n = 6) at the Atomic Centre of the Argentinean National Commission of Atomic Energy (CNEA) with a minimum and maximum dose of 0.32 - 0.41 kGy. Twenty gram of each muscle from Gt and Gc were taken at 24 h, 7 days, 14 days and 21 days post-irradiation, and all samples were individually processed by means of artificial digestion. The ML were then inoculated into mice to evaluate the intestinal phase and the muscular phase. All recovered larvae from Gt and Gc samples showed integrity of the cuticle and active motility. Adult worms and ML were recovered from all mice inoculated with ML from Gc. However, no adult worms or ML were obtained in mice inoculated with ML from Gt. These results show the efficacy of irradiation to inhibit the development of T. pseudospiralis and T. spiralis in the host, which could possibly be extended to other non-encapsulated species, but further studies are needed to demonstrate this hypothesis.

Músculo romboides supraescapular de la Llama (Lama glamá) / Rhomboideus suprascapularis muscle of the llama (Lama glama)

El músculo romboides (m. rhomboideus) forma parte de la sinsarcosis que une la cintura del miembro torácico con el esqueleto axil en los mamíferos domésticos. En estas especies, está integrado por los músculos romboides cervical (m. rhomboideus cervicis) y romboides torácico (m. rhomboideus thoracis), siendo imposible establecer el límite entre ellos, a diferencia de lo que sucede en el Hombre. Los carnívoros en general, el cerdo y el conejo presentan, además de las partes mencionadas, el músculo romboides de la cabeza (m. rhomboideus capitis). En la llama, la porción cefálica está ausente y la cervical pobremente desarrollada. Los autores proponen sumar a las porciones cervical y torácica del músculo romboides de este camélido sudamericano, al músculo romboides supraescapular (m. rhomboideus suprsacapularis), descrito por Lesbre (1903), en el camello y el dromedario. En este trabajo se establecen las inserciones, dimensiones e inervación del músculo romboides supraescapular de la llama, elementos que permiten definirlo como otra porción del complejo muscular romboideo en dicha especie. Además, se postula su acción como elevador de la escápula, dirigiéndola craneal y dorsalmente.

The rhomboideus muscle of the domestic mammals is part of the muscular set that joins the scapular waist to the axial skeleton. In these animals, the forenamed muscle has a cervical portion (m. rhomboideus cervicicis), and a thoracic portion (m. rhomboideus thoracis). Unlike in the man, these parts cannot be separated. In addition, carnivores, pigs and rabbits also have a cephalic portion (m. rhomboideus capitis). In the llamas, the cephalic portion is absent, and the cervical part is poorly developed. The authors propose to add a suprascapular portion (m. rhomboideus suparsacapularis), first described by Lesbre, 1903 in camels, to the Rhomboideus muscular complex of the llama. In this study, the authors describe the length, insertions, and inervation of the Rhomboideus Suprascapular muscle of the llama in order to define it as another portion of the Rhomboideus muscular complex. Moreover, the forenamed muscle is proposed as a scapular elevator.

Circuito venoso cervical de la llama (Lama glama) / Cervical venous circuit of the llama (Lama glama)

La particular estructura de la región cervical de la llama (Lama glama) se traduce en numerosas diferencias anátomo-funcionales respecto de otros mamíferos domésticos. Entre ellas merece destacarse un distintivo circuito venoso, cuya descripción es el objetivo de este trabajo. Para el presente estudio, se utilizaron diez llamas, machos, adultos y de aptitud lanera. Para resaltar las venas del cuello se procedió a la repleción de las mismas y, posteriormente, se disecaron los vasos utilizando técnicas e instrumental convencional. Se describe la morfología particular de un circuito venoso, en el cual las venas yugular externa, vertebral e intervertebrales son las principales protagonistas. El hecho destacado radica en las anastomosis establecidas entre las citadas venas, uniones dispuestas en forma metamérica, formando un circuito o by pass yugular-vertebral. La existencia de comunicaciones o shunts venosos, permite definir un circuito de circulación colateral derivando la sangre principalmente hacia la vena yugular externa. La longitud del cuello y los hábitos de pastoreo indican que esta disposición vascular es un hecho facilitador del retorno venoso, en cuanto a su dinámica y al control regional del flujo sanguíneo.

The neck region of the Llama (Lama glama) has several structural and functional particularities in comparison to the same region of other domestic mammals. Among them, it is noticeable a distinctive venous circuit whose description is the purpose of this study. In order to perform the study, the veins of the neck of ten adult, male, wool type llamas were dissected with conventional technique. Previously to dissection, the veins were filled with a repletive substance. The authors describe the morphology of a venous circuit in which external jugular vein (v. jugularis externa), vertebral vein (v. vertebralis), and intervertebral veins (vv. Intervertebrales) are the main participating vessels. The distinctive feature is the fact that the metameric anastomoses between the forenamed veins leads to a jugular-vertebral by pass. The existence of venous shunts provides a circuit of collateral circulation, diverting the blood mainly towards the external jugular vein. Grazing habits and the length of the neck suggest that the vascular structure previously described facilitates the venous return, contributing to the regional blood stream control.