(235) Edited Steroid Five Alpha-Reductase 2 Promoter and Boar Development

Trish Berger¹, Barbara Jean Nitta-Oda¹, Insung Park¹, Xiang Yang¹ and Yanhong Liu¹

1. Department of Animal Science, University of California Davis, Davis, California

Abstract Text:

Founder pigs with deletions in the promoter region of steroid five alpha reductase type II (SRD5A2) were created using CRISPR/Cas9 technology. Oocytes were aspirated from ovaries retrieved at a slaughterhouse, in vitro matured, in vitro fertilized with a mixture of Yorkshire and Berkshire semen, and the designed guide and Cas9 electroporated into zygotes. Edited and unedited embryos were transferred to the oviduct at approximately the four-cell stage. A recipient farrowed four unedited pigs, an edited male and an edited female. Founders contained no wildtype sequence but were mosaic for specific edits in the promoter region. Coat color indicated the male founder was sired by the Berkshire and the female founder by the Yorkshire.  Both had some Duroc ancestry based on coat color of their offspring and the relatedness of the oocyte donors is unknown. Founders were bred to each other to produce experimental animals with disrupted DNA sequence in the SRD5A2 promoter region. Six control boars were randomly selected from available litters that contained some Duroc, Berkshire, or Hampshire ancestry. Experimental animals were characterized by normal external genitalia. Growth of the seven experimental boars was comparable to that of control boars, as determined by weekly body weights.  All animals appeared healthy as assessed by blood chemistry screens at 18 weeks of age. Organs were collected and weighed at 6 months of age. Bulbourethral glands from the seven edited boars were 25% smaller than those from the six control boars at 6 months of age (p < 0.05). Weights of testes, prostates, and seminal vesicles varied among individual boars but organs from control and edited boars were in the same range of weights. The mRNA for SRD5A2 was not detected in five of the seven edited boars. Prostates exhibited minimal SRD5A2 mRNA response to edits and comprise a quite small proportion of the male reproductive tract. The seminal vesicles and bulbourethral glands comprise a significant proportion of the tissues capable of modifying steroids. An estimated eight-fold decrease in SRD5A2 expression was observed in seminal vesicles from edited boars ( P < 0.10); this is potentially relevant to the overall SRD5A2 response. In contrast to the decreased growth of the bulbourethral glands in the experimental animals, weights of heart, lung, spleen, liver, and kidneys did not differ between the experimental boars and the control boars. No apparent abnormalities in organ function were detected. These results indicate that disruption of the SRD5A2 promoter sequence is compatible with overall animal health, a prerequisite if this approach is to be used commercially in a strategy to eliminate boar taint while maintaining the favorable growth characteristics of intact boars.

(Funding for initial and subsequent studies include UC Davis Academic Senate New Initiatives, USDA NIFA Exploratory Grant 2019-67030-29097, a UC Davis Clear Center award from National Pork Board, and a donation from the California Pork Producers Association.)