One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

PSIX-26 Effect of enhanced nutrition during early life on the transcriptional profile of the arcuate nucleus region of the hypothalamus in Holstein Friesian bull calves

Improved nutrition and metabolic status during early life are known to advance sexual development in the bull calf. This is mediated through complex neuroendocrine signaling at the level of the hypothalamic-pituitary-testicular (HPT) axis resulting in precocious testicular development and spermatogenesis. The arcuate nucleus region (ARC) of the hypothalamus is centrally involved in the integration of peripheral metabolic cues with GnRH neuronal function and gonadotropin pulsatility which in turn stimulate testicular development. However, the precise molecular mechanisms regulating the influence of prevailing nutritional status on hypothalamic function are yet to be fully elucidated. The objective of this study was to evaluate differentially expressed genes (DEG) within the ARC of bull calves offered contrasting planes of nutrition during the first 12 weeks of life, to coincide with the characteristic transient gonadotropin rise. Holstein-Friesian bull calves with a mean (+SD) bodyweight and age of 48.8(5.3) kg and 17.5(2.8) days, respectively, were assigned to either a high energy (H; n = 15) or moderate energy (M; n = 15) plane of nutrition designed for growth rates of 1.0 and 0.5 kg/day, respectively. At 12 weeks of age all calves were euthanized and the ARC harvested. RNA was isolated from all ARC samples and RNAseq analysis conducted. As expected, H calves were heavier at euthanasia (H=112 kg; M=88 kg, P < 0.001). RNAseq analysis resulted in the identification of 83 DEG (P.adj< 0.1; fold change >1.5), all of which were down-regulated in H compared to M calves. DEG were enriched for biological pathways associated with immune function including complement system and acute phase response signaling (P.adj< 0.01). However, no DEG identified were directly involved in reproductive developmental processes. Results from this study suggest that the typically positive effect of enhanced nutrition on reproductive development may not be apparent within the ARC at 12 weeks of age.

PSX-32 Late-Breaking Abstract: Production of a Gene Knock-In Bull Calf by Embryo-Mediated Genome Editing

Genome editing offers an opportunity to introduce targeted gene insertions into livestock breeding programs. Molecular geneticists have typically employed a donor repair template and the homologous recombination (HR) pathway in somatic cells to introduce gene knock-ins into livestock genomes, followed by cloning. Editing embryos directly to achieve targeted gene knock-ins is inefficient, especially for introducing large DNA sequences. Here we report using a one-step method to produce a gene knock-in bull calf by cytoplasmic microinjection of CRISPR/Cas9 reagents into a bovine embryo. In vitro fertilized one-cell bovine zygotes were injected with a gRNA/Cas9 ribonucleoprotein complex and homology mediated end joining donor template containing the sex determining region Y (SRY) gene, the green fluorescent protein (gfp) reporter gene driven by the SV40 promoter, and one kilobase homology arms targeting the H11 safe harbor locus on bovine chromosome 17. Seven-day blastocysts were evaluated using fluorescent microscopy, and nine green fluorescent embryos were transferred to synchronized recipients. Ultrasound evaluation at 35 days revealed one pregnancy. In April 2020, a healthy 50 kg male calf was born. DNA was extracted from placenta, blood and a fibroblast line derived from the calf and analyzed for SRY-GFP knock-in, as well as genotypic sex. PCR and Sanger sequencing revealed the biallelic edit of the target location on chromosome 17, with the insertion of three or seven copies of the SRY-GFP construct in addition to donor plasmid backbone, or a 26 base pair insertion, and an XY genotype. Future analysis of the XX offspring inheriting the SRY gene on chromosome 17 from this knock-in bull will reveal whether inheritance of the bovine SRY gene is sufficient to trigger the male developmental pathway in cattle.

PSVII-41 Late-Breaking Abstract: Impact of enhanced early life nutrition on the testes transcriptional profile of the bull calf

Enhanced plane of nutrition and metabolic status are known to progress sexual development in the bull calf. This is facilitated through neuroendocrine signaling in the hypothalamic-pituitary-testicular signaling axis. Improved nutrition may lead to early release of hypothalamic gonadotropin releasing hormone (GnRH) and the subsequent production of follicle stimulating luteinizing hormones in the anterior pituitary and ultimately development of the testes and sexual development. Nonetheless the precise molecular mechanisms leading to this effect are yet to be expounded. The purpose of this study was to evaluate differentially expressed (DE) genes within the testes of bull calves differentially fed for the first 12 weeks of life. The main functions of the testes are the biosynthesis of androgens by the leydig cells and sperm production by the seminiferous tubules. Accelerating the development of this tissue may lead to enhanced reproductive development at an earlier stage. Holstein Friesian bull calves with a mean (SEM) age and bodyweight of 17.5 (2.8) days and 48.8 (5.3) kg, respectively, were assigned to either a high (H; n = 15) or moderate (M; n = 15) plane of nutrition, to achieve an average target growth rate of 1.0 and 0.5 kg/day, respectively. Calves on H and M received 1.5 and 0.5 kg of milk replacer (MR) per day, reconstituted at 15 and 12.5% (w/v), respectively. Calves on H were offered concentrate ad libitum, while those on M received, 500g day-1. Both groups were offered 500 g of hay daily. At 87 days (±2.141) of age, all calves were euthanized and the testes recovered from all calves. RNA was isolated from all testes samples and subsequently subjected to RNAseq analysis. Calves offered a higher plane of nutrition were heavier at slaughter (112 v 88 kg, P < 0.001), reflective of their higher ADG (0.88 v 0.58 kg, P < 0.001). Similarly, H also had heavier testes (29.2 v 20.1 g, P < 0.05) compared to M calves. Results from RNAseq analysis identified 27 DE genes (p.adj< 0.1; fold change >1.5). Cholesterol biosynthesis was an enriched pathway and reproductive system development was also affected. We also saw that Claudin11 was an up regulated DE gene which is involved in sertoli cell development. The results from this study would advocate that there is potential effects of improved nutrition on reproductive function within the testes of bull calves at 12 weeks of age.

Correction to: Management of atresia ani type II in a day-old Bunaji bull-calf

The initial version of the article published in Volume 17 number 2 (June, 2019) contained errors in the name of A Agbonu and his affiliation. The correct name is OA Agbonu, while correct affiliation is Veterinary Physiology and Biochemistry, University of Abuja, Nigeria.

Welfare implications for dairy bull calves.

Dairy bull calves may experience compromised welfare as a result of production practices and management techniques implemented on farm. This includes high disease incidence due to poor ventilation, pain experienced as a by-product of processing procedures, social isolation, and hunger from inappropriate nutrition. Although dairy heifer calves may experience similar issues, these conditions are often exacerbated for dairy bull calves due to the low economic value of the individual animal. In addition, given the bull calf will likely not remain on the farm, this group of animals are more likely to receive less adequate care in the first weeks of life. Welfare issues such as increased rates of dystocia, failure of passive transport, dehorning, castration, and long transportation distances are all critical and will be discussed in this review. Therefore, the objectives of this article are to (1) evaluate current welfare concerns specific to dairy bull calves, (2) identify areas for improvement to mitigate poor welfare outcomes, and (3) review proper euthanasia techniques and protocols specific for calves. Ultimately there is still much to learn about specific areas for improvement relating to the welfare of dairy bull calves and future studies are needed. However, the industry should properly manage the welfare challenges of bull calves, identify opportunities within the industry to increase their value, and uphold our ethical responsibility to these animals.

Spontaneous Appearance and Transmission of Polydactyly in Dexter Cattle

A 3-yr-old Dexter cow and her yearling Dexter heifer calf exhibited polydactyly. Neither animal was linebred within 5 generations. This cow-calf pair represented the first reported occurrence of polydactyly in Dexter cattle in the US or abroad. Based upon external examination, the cow was classified as having a spontaneous unilateral case of polydactyly with an extra digit along the medial digit of the right front limb and the heifer was classified as having bilateral polydactyly because both front limbs exhibited an extra digit along the medial digit. Radiographic examination confirmed bilateral status of the heifer and revealed bilateral status of the cow. The front feet of the cow and heifer had extra bone formation consistent with an extra digit along the medial digit. Neither animal suffered from limited mobility to date or required hoof treatments. The cow produced a second calf from a different sire, a bull calf that did not appear polydactylous per external examination and was not examined radiographically. The two polydactylous animals will remain in the breeding herd to produce more study calves unless their fitness becomes compromised. Genetic aspects of the cases are discussed.