Archaeological evidence from the last 8,000 years indicates that early African food-producing societies relied predominantly on livestock, particularly cattle, rather than crops. Cattle have since remained central to economies ranging from sedentary farming to mobile pastoralism across diverse ecosystems. The widespread use and persistence of cattle across Africa likely reflects their admixed ancestry, combining African taurine (Bos taurus), indicine (Bos indicus), and European taurine lineages, which were introduced at different times over the last ~8,000 years. To determine how varied human livelihood patterns and environmental factors affected the geographic distribution of these ancestries, we generated whole-genome sequences from 1,392 African cattle within a combined dataset of ~4,900 genomes, including two ancient genomes, one from Great Zimbabwe (~750 years old) and one from the Western Cape (~280 years old). Using local ancestry inference, we show that taurine and indicine lineages, despite co-existing in the same genomes for over a millennium, have followed markedly different trajectories: taurine ancestry is strongly structured geographically, maintained by sedentary communities under local selection; indicine ancestry is poorly geographically structured which likely reflects dispersal through pastoralist movement; and European taurine heritage is highly localised in South Africa where it is associated with strong selection for beef cattle. Together, these results reframe African cattle as carriers of co-resident lineages, each preserving a distinct record of human subsistence strategy, ecological constraint, and adaptive evolution.

Background The Mediterranean indigenous Baladi cattle is known for its exceptional adaptability to harsh environment, making it a prime candidate for sustainable grazing systems amid global climate change. This study investigates the ancestors of the Baladi genome, genomic regions under selection, and adaptation mechanisms of Baladi cattle. Results A unique balanced global admixture of taurine and indicine genomes was found, composed of Indicine, European taurine and African taurine ancestors. A total of 103 chromosomal regions with significant local deviation were found, taken as selection signatures. A clear tendency of selection for the Indicine and African taurine ancestors at the expense of European taurine was observed. Bioinformatics analysis of the selection signature regions revealed genes candidates by location and function to affect Baladi adaptive selection. Conclusions The Baladi genome is a unique rich mosaic of three domesticated bovine sub-specie lineages. Thus, the Baladi cattle is a mixture already sieved for adaptation to the harsh Mediterranean climate. These findings emphasize the urgent need for conservation efforts to preserve the genetic diversity and adaptive traits of this valuable, endangered breed.

Abstract Tall fescue pastures support 40% of U.S. cow–calf herds, but fescue toxicosis causes $2 billion in annual losses. Management strategies often rely on genetics to reduce heat stress under toxicosis. In this ongoing project, we evaluated a gene editing approach to introduce the slick mutation (mutation in the prolactin receptor that gives cattle a sleek short hair) in Angus cattle grazing toxic fescue. We hypothesized that calves inheriting slick mutation have similar development to wild-type calves up to weaning but greater weight gain when grazing toxic fescue in the stocker phase. Multiparous Angus cows (n = 222) were synchronized and artificially inseminated with the semen of an Angus bull submitted to CRISPR-Cas9 edition to carry the slick mutation mosaic (Aa allele). 75% of the offspring were expected to be slick and 25% expected to be wild-type. Calves born within the first 30 days of the calving season (n = 94) were monitored for behavior. At birth coat color, sex, hair length in the scapula and shoulder to rump length (length from neck–shoulder junction to the end of the tailhead, following the spine) were recorded. In a subgroup (n = 13), the time to stand and time to nurse were measured as indicators of calf vigor, defined respectively as the interval from complete expulsion of the calf to standing on all four limbs for ≥ 5 consecutive seconds and to the first successful suckling. Within 20-30 days after birth thermal drone imaging (EVO II Dual 640T V3 manual) was conducted to assess surface temperature and 4 experience operators watched the behaviors of each calf for 30 minutes from 12 pm to 3:30 pm. The following behaviors in the 30 minutes interval were analyzed: time in the shade, time in the sun, time walking, time laying, and time nursing. Statistical analyses were performed in GraphPad Prism using simple linear regression. Hair length was not associated with birth weight, time to nurse, shoulder-to-rump length, or surface temperature (P > 0.10). However, hair length showed a positive linear association with time to stand (P = 0.024; R²=0.59). For each unit of increase in hair length the time to stand increase in 27.3 times. For behavior variables, there was no association between hair length and time in the shade, time in the sun, time walking, time laying down, or time nursing (P > 0.10). In conclusion, during the first 30 days of life we detected no significant effects of slick mutation in the calf behavior and development in agreement with our hypothesis. By the time this abstract will be presented we will have more information of calf development to share and genotype results to assure which ones are slick and wild-type.

Kisspeptin knockout (KISS1-/-) pigs exhibit hypogonadotropic hypogonadism. Hormone analogs targeting different levels of the hypothalamic-pituitary-gonad axis were used to characterize the secretion of reproductive hormones (LH, luteinizing hormone; and follicle-stimulating hormone, FSH) and ovarian responses (estradiol and progesterone) in KISS1-/- gilts. Uteri and ovaries were collected from KISS1+/+ and KISS1-/- gilts to confirm ovulatory outcomes. Pulses of LH and FSH were observed in KISS1-/- gilts that differed (P < 0.05) in amplitude and nadir from pulses in wild-type KISS1+/+ and KISS1+/- gilts. A neurokinin B (NKB) agonist stimulated LH but not FSH in wild-type gilts, whereas NKB affected neither LH nor FSH in KISS1-/- gilts. The kisspeptin receptor agonist, C6, stimulated LH secretion in wild-type gilts but not in KISS1-/- gilts (P < 0.05). Secretion of LH in KISS1-/- gilts depends on the dose and frequency of gonadotropin-releasing hormone (GnRH). Priming with estradiol-17β and GnRH before pregnant mare serum gonadotropin and human chorionic gonadotropin treatment resulted in luteal structures on the ovary of KISS1-/- gilts, though ovulation rate was less (P < 0.05) than KISS1+/+ gilts. This is the first report showing NKB regulation of LH secretion in swine. It is confirmed that a single copy of the KISS1 allele in gilts confers normal gonadotropin secretion following stimulation with NKB, kisspeptin, and GnRH analogs. The sustained activation of the kisspeptin receptor by C6 generated long-lasting LH secretion in gilts to induce ovulation. Ovulation in KISS1-/- gilts is possible, but treatments need optimization to maximize ovulation rate.

A precision genome edit in the bovine CD46 gene (A82LPTFS87) dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned heifer. However, pathogen evolution threatens the long-term efficacy of such interventions. Here, our aim is two-fold: first, to determine whether BVDV can adapt in vitro to use the edited CD46 receptor to infect Madin–Darby bovine kidney (MDBK) cells, and second, to evaluate the ex vivo infectivity of culture-adapted viruses in cells from the CD46-edited heifer. Serial passage of BVDV on CD46-edited MDBK cells selected for virus variants capable of CD46-independent infection. Virus genome sequencing revealed mutations in the viral ERNS gene predicted to enhance HS-mediated entry. HS adaptation was confirmed by inhibiting virus infection with heparin or Heparinase I/III treatment. A naturally occurring HS-adapted field isolate from a persistently infected calf showed similar results. However, when tested on primary cells from the CD46-edited heifer, HS-adapted viruses showed reduced infectivity in skin fibroblasts, monocytes, and lymphocytes in a manner that correlated with HS expression. Thus, although BVDV can adapt to use HS as an alternative entry receptor, HS adaptation does not overcome the protection conferred by the CD46 edit in all relevant cell types.

Technological advances in genomics and bioinformatics made it possible to study the genetic structure of breeds and understand genome changes caused by selection over generations. Our objective was to evaluate selection signatures (SS) in Nelore, Gir, and Red Sindhi cattle from Brazil and the Asian continent to identify divergent variants due to the history of formation and selection of populations, with a focus on the SS of animals from Brazil. Extended haplotype homozygosities between populations (XP-EHH), the ratio of site-specific extended haplotype homozygosity between populations (Rsb), and the allelic fixation index (Fst) were used to detect SS. Considering a window size of 50-kb, a non-sliding window approach was used to define SS regions. A total of 62, 57, and 72 genes were co-located within SS regions for Nelore, Gir, and Red Sindhi, respectively, and used to perform functional analyses per breed. Most genes were associated with productive and reproductive traits, while others were related to thermotolerance, the immune system, temperament, and coat color. The identified SS demonstrate how animal breeding programs shape the genetic makeup of these breeds to meet production system requirements, given that animals from Brazil and the Asian continent have undergone different selection processes. The identification of genes related to thermotolerance, temperament, and the immune system suggests specific alleles have enabled animals to adapt to environmental conditions and selection criteria in Brazil. Understanding SS can support breeding strategies for Nelore, Gir, and Red Sindhi cattle, contributing to enhanced resistance, adaptation, and productivity to meet food production demands.

Bovine viral diarrhea virus (BVDV) infection during pregnancy is a significant contributor to reproductive failures in cattle. The bovine receptor for BVDV (CD46) was previously edited with a six amino acid substitution (G82QVLAL to A82LPTFS) and shown to have significantly reduced BVDV susceptibility in a Gir heifer calf. Since a role for CD46 has been proposed in mammalian fertilization, our objective was to assess the edited heifer's fertilization rates, early embryonic development, and germline transmission conformation of the edit. Cumulus oocyte complexes were collected from the edited heifer and unedited females, fertilized with semen from an unedited bull and cultured until the blastocyst stage. Ultrasound examinations and serum progesterone concentration were also monitored to confirm estrous cyclicity in the CD46-edited heifer. Estrous cyclicity was normal with visualization of a corpus luteum and elevated progesterone concentrations. Fertilization rates and blastocyst development were not different in oocytes from edited and unedited controls. Genome sequence analysis of blastocysts confirmed germline transmission of either edited allele from the heifer. Subsequently, the CD46-edited heifer was artificially inseminated with semen from an unedited Gir bull and fertility status was confirmed with a diagnosed conception at Day 35 of gestation. Thus, a six amino acid substitution in CD46 did not negatively affect fertilization of edited oocytes or early embryonic development when fertilized with semen from an unedited bull. An edited bull is still needed to similarly evaluate reproductive function of sperm cells carrying this CD46 edit.

CD163 is the primary receptor for PRRSV, and its SRCR5 domain, encoded by exon 7, is crucial for supporting PRRSV infection. Previous studies have used CRISPR/Cas9 technology to remove exon 7 from the host genome, and the edited pigs were completely resistant to PRRSV infection. In this study, we used CRISPR/Cas9 technology mimicking an adenine base editor (ABE) to edit the splice acceptor site of exon 7, rendering it nonfunctional. This alteration was intended to cause exon 6 to join directly to exon 8 during mRNA processing, resulting in a mature mRNA transcript that lacks exon 7, which encodes the SRCR5 domain. Piglets carrying the exon 7 splice site modification (CD163Ex7-ABE) were successfully generated. However, these pigs remained fully susceptible to infection with a PRRSV-2 isolate. Analysis of CD163 mRNA from the CD163Ex7-ABE pigs revealed that they predominantly expressed a mature CD163 mRNA lacking exon 7. However, due to cryptic splice sites, two additional mRNA isoforms were expressed, including an in-frame variant containing all of exon 7 and an extra 48 base pairs. This likely resulted in the expression of a full-length CD163 with a 16-amino-acid insertion upstream of the SRCR5 domain, which was sufficient to render the animals susceptible to PRRSV. Overall, our results demonstrate that merely modifying the splice acceptor site of CD163 exon 7 is not sufficient to generate PRRSV-resistant pigs.

Genome editing is the latest breeding tool capable of accelerating the rate of genetic improvement for health and well-being traits in food animals. It enables the introduction of beneficial alleles within a single generation, including those of low frequency or absent in the population, while effectively bypassing linkage drag. For the dairy industry, genome editing can be used to make rapid genetic improvements that are precise, efficient, and transgene-free for functional traits that are not practically addressed without disrupting conventional breeding goals for overall economic merit based on genomic selection. Herein, various case studies for dairy cattle breeding are presented that demonstrate applications of genome editing for enhancing heat stress tolerance, reduced disease susceptibility, and/or other qualitative traits absent in some breeds. One case highlights the success of simultaneous editing of multiple loci through recent advancements in embryonic stem cell biology. Multiplexed editing is crucial for addressing the polygenic nature inherent to many economically important traits in livestock. However, maximizing the benefits of genome editing depends on the continued discovery of targets for editing that are commercially important. Commercialization also depends on rapidly evolving regulatory statutes for risk assessment, where some countries already permit the commercialization of cattle with non-GMO genome alterations through existing regulations. New breeding technologies like genome editing are now poised to have significant impact in equipping elite performance cattle to be more resilient to infectious disease and climate change without the loss of production gains obtained from decades of selection.

The increasing demand for natural products is reshaping meat consumption, with grass-fed and grass-finished beef emerging as a lucrative option. However, shifting from conventional grain-fed style to grass-fed systems necessitates numerous adjustments in management decision-making and practical operations. To meet the knowledge gap, our study delved into various OMIC data, including growth performance, microbiome, metabolites, and epigenetics in a closed Wye Angus herd. Our findings revealed no difference in meat tenderness between the two treatments (P = 0.25), but significant diet effects on epigenetics with a substantial number of DNA methylation marks, which are of paramount importance. Moreover, we ascertained the microbiome in intestinal and rumen after weaning. Grass-fed cattle exhibited a significantly greater level of microbial diversity. The top 20 essential genera identified with random forest analysis could serve as microbial biomarkers and associate with the components of bile acids. Notably, the metabolomic analysis unveiled changes in glucose availability and utilization, variations in free fatty acids and carnitine-conjugated lipids, and altered β-oxidation, which influence complex lipid hydrolysis, contributing to the accumulation of anti-inflammatory n3 polyunsaturated fatty acids in grass-finished cattle. At the same time, greater concentrations of n6 PUFAs in grain-finished animals could promote inflammation and oxidative stress. With the discovery of low cortisol levels in grass-fed cattle, the cattle have health and welfare benefits in a grass-fed manner. Most importantly, they also provide fresh insights into microbial interactions in the rumen under different feed schemes and their ecophysiological implications, further aiding in developing rumen manipulation strategies to enhance feed conversion ratios and methane emission.

Criollo cattle, the descendants of animals brought by Iberian colonists to the Americas, have been the subject of natural and human-mediated selection in novel tropical agroecological zones for centuries. Consequently, these breeds have evolved distinct characteristics such as resistance to diseases and exceptional heat tolerance. In addition to European taurine (Bos taurus) ancestry, it has been proposed that gene flow from African taurine and Asian indicine (Bos indicus) cattle has shaped the ancestry of Criollo cattle. In this study, we analysed Criollo breeds from Colombia and Venezuela using whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) array data to examine population structure and admixture at high resolution. Analysis of genetic structure and ancestry components provided evidence for African taurine and Asian indicine admixture in Criollo cattle. In addition, using WGS data, we detected selection signatures associated with a myriad of adaptive traits, revealing genes linked to thermotolerance, reproduction, fertility, immunity and distinct coat and skin coloration traits. This study underscores the remarkable adaptability of Criollo cattle and highlights the genetic richness and potential of these breeds in the face of climate change, habitat flux and disease challenges. Further research is warranted to leverage these findings for more effective and sustainable cattle breeding programmes.

Following infection of a porcine dam with PRRSV around 90 days of gestation, the virus crosses the placenta and starts to infect fetuses. This can lead to consequences such as abortions, stillbirths, and respiratory issues in newborn piglets. CD163 is an essential cellular viral entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serinethreonine (PST) domains. Gene-edited pigs possessing a complete deletion of CD163 are resistant to PRRSV infection. Recently, we demonstrated that pigs harboring a clean deletion of CD163 exon 13 (Delta Exon13 CD163 pigs) which encodes the first 12 amino acids of the CD163 PSTII domain were not susceptible to PRRSV infection. In this study, Delta Exon13 CD163 (-/-) gilts were bred with wildtype CD163 (+/+) boars producing heterozygous, CD163 (+/-) fetuses. We found that fetuses with a wildtype CD163, recovered between day 103 of gestation or 17 days after the maternal infection with PRRSV, were fully protected from PRRSV in dams containing a clean deletion of CD163 exon 13. These findings suggest a feasible approach for eliminating PRRSV-related reproductive illness, which is a significant cause of economic losses in agriculture.

Sustainable improvement of production in most tropical dairy systems is a significant challenge, because the cattle breeds with the most genetic potential for milk output relative to native tropical breeds have not been selected for these types of environments. Multiplex genome editing provides a potential solution to introduce beneficial sequence variants (SVs) into elite animals for tropical adaptation in a single generation. Bovine sequence variants for heat tolerance, stature, milk yield, and disease-tolerance traits were identified and genotyped across indigenous African, dairy Zebu, and dairy Taurine breeds to validate those targets best suited for introgression by genome editing. In vitro fertilized embryos from a series of matings were used to produce embryonic stem cells (ESCs) and were subsequently multiplexed edited prior to cloning by somatic cell nuclear transfer. A set of best target SVs for genome editing was established for the Holstein and Gir breeds. ESCs were produced and cloned following treatment and validation screening for multiplex alterations of up to four target genes. Currently, 12 animals have been born, and all the mature males have produced viable semen that will be submitted for regulatory review in a series of countries in Sub-Saharan Africa and South America. Multiplex genome editing based on homology-directed repair combined with cloning of bovine ESCs provides an opportunity to initiate genetic improvement of polygenic traits in cattle. Combining genomics and genome editing provides new opportunities to breed more resilient dairy animals for the tropics that should improve animal and farmer livelihoods.

CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16 amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main physiological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection.

Global warming is a major challenge to the sustainable and humane production of food because of the increased risk of livestock to heat stress. Here, the example of the prolactin receptor (PRLR) gene is used to demonstrate how gene editing can increase the resistance of cattle to heat stress by the introduction of mutations conferring thermotolerance. Several cattle populations in South and Central America possess natural mutations in PRLR that result in affected animals having short hair and being thermotolerant. CRISPR/Cas9 technology was used to introduce variants of PRLR in two thermosensitive breeds of cattle - Angus and Jersey. Gene-edited animals exhibited superior ability to regulate vaginal temperature (heifers) and rectal temperature (bulls) compared to animals that were not gene-edited. Moreover, gene-edited animals exhibited superior growth characteristics and had larger scrotal circumference. There was no evidence for deleterious effects of the mutation on carcass characteristics or male reproductive function. These results indicate the potential for reducing heat stress in relevant environments to enhance cattle productivity.

Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 days of age, n = 42; gilts to 160 days of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues were collected for gross morphology and histology, and a fat biopsy was collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1+/+ and heterozygote KISS1+/- animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1-/- boars and gilts compared with KISS1+/+ and KISS1+/- animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1-/- boars than in KISS1+/+ and KISS1+/- boars (P < 0.05). Hypogonadism was present in all KISS1-/- gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Sheep is an important livestock species raised globally to pro-duce meat,milk,wool,and other by-products.During the Neolithic Revolution,sheep were domesticated in the Fertile Crescent of Southwest Asia around 10,000 years ago(Chessa et al.,2009).Nat-ural and artificial selection has been driving the development of various sheep breeds/ecotypes adapted to different climatic and production conditions.Sheep breeds often differ in coat color,pres-ence or absence of horns,tail shape,etc.Sheep tails can be divided into five main patterns,including fat-long tail,fat-short tail,thin-long tail,thin-short tail,and fat rump(i.e.,'tailless'),which are determined by the length and number of caudal vertebrae as well as the amount of fat deposition.In modern breeding programs,the thin-short tail is considered preferable since other tail patterns have specific issues of concern.For example,fat tails are indicated to be associated with low mating success,animal locomotion,excessive fat deposition,increased production costs,and reduced consumer preference.Similarly,thin long-tailed sheep require tail docking as part of the an-imal management practice,which is a concern for animal welfare.