Karim Khalil

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

The myostatin (MSTN) gene is important because of its role in regulation of skeletal muscle growth in all vertebrates. In this study, CRISPR/Cas9 was utilized to successfully target the channel catfish, Ictalurus punctatus, muscle suppressor gene MSTN. CRISPR/Cas9 induced high rates (88-100%) of mutagenesis in the target protein-encoding sites of MSTN. MSTN-edited fry had more muscle cells (p < 0.001) than controls, and the mean body weight of gene-edited fry increased by 29.7%. The nucleic acid alignment of the mutated sequences against the wild-type sequence revealed multiple insertions and deletions. These results demonstrate that CRISPR/Cas9 is a highly efficient tool for editing the channel catfish genome, and opens ways for facilitating channel catfish genetic enhancement and functional genomics. This approach may produce growth-enhanced channel catfish and increase productivity.

Keywords: Angioarchitecture, Testes, Testicular artery, Pampiniform plexus, Thermoregulation.

Modern experimental and surgical approaches as well as normal physiological Studies of the blood supply of the mammalian testis are increased in order to The vascular patterns of testis and epididymis reflects on the thermoregulatory mechanism and this in turn on spermatogenesis which is normally known to exist at a temperature lower than that of the body (extra-abdominal testes) in all animals except marine mammals and birds which have intra-abdominal testes. The testicular arteries are the main source of blood supply of the testis and epididymis. They are two slender vessels of considerable size; arise from the abdominal aorta. The course of the testicular artery among the different species of animals shows considerable species variations especially in its architectural pattern, distribution as well as the mode of its termination. Topographically, Testicular artery can be divided into three parts (abdominal, funicular and marginal parts) along its course. The coils of the funicular part of the testicular artery forming a cone-like structure with its base fixed to the head of the testis. The bulk of the cone is built of several arterial loops, venous network of pampiniform plexus as well as the spermatic fascia. The increase in the length of the testicular artery with increase in the size of the testes played a great role in the degree of complexity of the architectural vascular pattern. The degree of complexity is affected by the number of coils formed by the vessel. The testicular artery appears more complex in species which have high temperature gradient between abdominal and testicular blood. We need more detailed comparison and accurate description on the architectural pattern of the testicular blood vessels and its relation with thermoregulation in different species of animals and comprehensive information about the morphmetric data of the testicular vessels.

ABSTRACT

(Key words: Angioarchitecture, Testicular artery, Testes)

The topography and morphometry of the testicular artery and pampiniform plexus, in the spermatic cord and testis were cited. 150 Samples were collected from different species of farm animals for this study. Different masses (Urographine®, Latex and Epoxy) were injected either through the testicular artery or the pampiniform plexus. The coils of the testicular artery forming a cone-like structure with its base fixed to the head of the gonad. The variations in the vascular Pattern of the testis among farm animals have showed a high degree of species specificity. The funicular part of the testicular artery gave off two epididymal branches to the head and tail of epididymis before it became marginal artery. The architectural pattern of the pars marginalis was classified into four patterns in farm animals. The mean values of the lengths, diameters and the degree of the vascular convolutions were recorded and statistically tabulated. The increase in the length of the testicular artery with increase in the size of the testes played a great role in the degree of complexity of the architectural vascular pattern among the species understudy. The degree of complexity is affected by the number of coils formed by the vessel. The testicular artery appears more complex in buffalo and ram than camel, donkey and rabbit. The pampiniform plexus originated from marginal plexus which gathered blood from the intraparenchymal network of veins. The coiling pattern of the artery and density of the veins in the pampiniform plexus was varied in different species of animals and played a major role in the heat regulation mechanism of the testis.

The aim of the study was to compare the pattern and density of the vascular layer of testis in some farm animals. The study was conducted on 50 specimens (10 for each species). The collected specimens are divided into two equal groups; the first group is used for gum milk latex injection while the other group is prepared for corrosive cast technique. The differences in the vascular pattern of the vascular tunic among the various species of farm animals are contributed to the architectural pattern of the marginal artery as well the mode of its termination so the current study suggest four patterns. The density of the tunica vasculosa in buffalo is pronounced in the distal third of both lateral and medial surfaces of the testis while it appears scanty in the proximal two third of both surfaces. In ram it is more pronounced than in buffalo while in camel the density of the tunica arteriosa testis is poor. In donkey the density of the tunica arteriosa testis is more pronounced in the distal two third of both surfaces of the testis. The density is poor and less pronounced in rabbit.