26 The Kisspeptin Knockout Pig: a New Model for Dissecting Mechanisms Controlling Reproduction
Authors: Lents Clay A , Florez Julio M , Martins Kyra , Bostrom Jonathan R , Rodriquez-Villamil Paula , Ongaratto Felipe , Huisman Abe , Larson Sabreena A , Ahern Daniel F , Ross Caitlin E , Elsken Dorothy H , Cushman Robert A , White Brett R , Sonstegard Tad S 
Published: November 2023

Abstract
The biological role of kisspeptin in the reproduction of swine has been far less studied than in other species. In the porcine hypothalamus, kisspeptin is coexpressed with neurokinin B and dynorphin in KNDy neurons. Although kisspeptin-stimulated secretion of gonadotropin hormones in gilts was established early, increased expression of hypothalamic KISS1 was only recently confirmed to be associated with puberty and cyclicity in pigs. Gene knockout (KO) models to study kisspeptin in livestock did not exist until TALEN gene editing technology was used to knockout KISS1R in pigs, resulting in a hypogonadotropic-hypogonadal (HH) phenotype that caused pubertal failure. Little is known about the reproductive physiology of KISS1R KO pigs, except that exogenous follicle-stimulating hormone and gonadotropin-releasing hormone only partially restored gonadal development in boars. To further explore the biological function of kisspeptin and its receptor in swine, we recently used CRISPR/cas9-ribonucleoproteins to knockout KISS1 in pigs (10.3389/fgene.2022.1078991). A total of 684 zygotes were injected resulting in 69 piglets with 58% bearing KISS1 KO alleles (KISS1-/-). The proportion of KISS1-/- alleles did not affect BW or ADG in these mosaic founder pigs. Two boars and 2 gilts with complete KISS1 KO exhibited HH and failed to become pubertal. Germ and Sertoli cells were disorganized in small seminiferous tubules. Mating mosaic F0 pigs (frequency of KISS1-/-, 35 to 88%) resulted in 170 F1 piglets with KISS1-/- alleles observed in a Mendelian frequency. These KISS1-/- pigs displayed normal BW growth during development but were also HH. Ovarian development of KISS1-/- gilts was arrested at a juvenile state, although antral follicles were present. The KISS1-/- boars lacked testicular development and had dramatically reduced concentrations of androgens and skatole, suggesting the lack of boar taint in their meat. Importantly, KISS1 and KISS1R are expressed in ovarian, uterine and testicular tissue; however, the direct role of kisspeptin in these reproductive organs remains unknown in livestock. These KISS1-/- pigs provide a unique animal model that opens new avenues for understanding the role of kisspeptin and gonadotropin hormones in regulation of reproductive development and function in pigs as well as livestock in general. These animals facilitate our ability to explore questions such as: 1) how KNDy neurons function and control gonadotropin pulses or 2) what role does KISS1 play in the development and function of the ovary, uterus and testis in domestic animals. Some of these questions have heretofore only been addressable with difficult surgical modifications, and therefore, KISS1-/- pigs represent an alternative to painful procedures in livestock research. Pursuing these fundamental studies is expected to lead to methods that restore fertility in KISS1-/- pigs. This, in turn, could provide new possibilities for the elimination of surgical castration from pork production. The USDA is an equal opportunity provider and employer.