Uncovering Novel Features of the Pc Locus in Horn Development from Gene-Edited Holstein Cattle by RNA-Sequencing Analysis.

The Polled Celtic (Pc) mutation locus is a genetically simple single mutation that is the best choice for breeding polled cattle using gene editing. However, the mechanism of the Pc locus for regulating horn development is unclear, so we used gene editing, somatic cell nuclear transfer and embryo transfer to obtain polled Holstein fetal bovine (gestation time 90 days) with a homozygous Pc insertion (gene-edited Holstein fetal bovine, EH) and the wild-type 90 days Holstein fetal bovine (WH) as controls. The hematoxylin-eosin (HE) staining results showed that, compared to the WH, the EH horn buds had no white keratinized projections or vacuolated keratinocytes and no thick nerve bundles under the dermal tissue. Furthermore, DNA sequencing results showed that the Pc locus was homozygously inserted into the fetal bovine genome. A total of 791 differentially expressed genes were identified by transcriptome sequencing analysis. Enrichment analysis and protein interaction analysis results of differentially expressed genes showed that abundant gene changes after Pc insertion were associated with the adhesion molecule regulation, actin expression, cytoskeletal deformation and keratin expression and keratinization. It was also noted that the results contained several genes that had been reported to be associated with the development of horn traits, such as RXFP2 and TWIST1. This study identified these changes for the first time and summarized them. The results suggested that the Pc mutant locus may inhibit neural crest cell EMT generation and keratin expression, leading to failures in neural crest cell migration and keratinization of the horn bud tissue, regulating the production of the polled phenotype.

Polledness in Argentinean Creole cattle, five centuries surviving.

Polledness has been shown to have autosomal Mendelian inheritance, with the polled locus being dominant to the horned locus. This trait was mapped to the BTA1 centromeric end in several breeds. One of the distinctive attributes of Creole cattle, such as the Argentinean Creole, is the presence of long, lyre-shaped horns. However, polled native animals were reported before the introduction of modern selected European breeds. Here, we studied the origin of the polled mutation, either independent or introgressed, in a Creole line from the Creole cattle founder group at the IIACS-INTA Leales Experimental Station (northwest Argentina). The study sample (65 animals: 26 horned and 39 polled) was genotyped using high-density SNP microarrays and three previously reported genetic markers (P202 ID , P80kb ID and PG ). A genome-wide association study, selection signatures, linkage disequilibrium analysis and copy number variations were used to detect the responsible region and the segregating haplotypes/alleles. The interval mapped in the Leales herd (1.23-2.13 Mb) overlapped with the region previously reported in several European cattle breeds, suggesting that the same locus could be segregating in this population. The previously reported variants PF and PG were not detected, thus dismissing the Holstein-Friesian and Nellore origins of the polled phenotype in this native breed. Conversely, the presence of the Celtic variant PC suggests an almost complete co-segregation. The cluster analysis rejected the hypothesis of recent introgression, which is compatible with the historical record of polled Creole cattle in northwest Argentina.

Sensitivity and wound healing after hot-iron disbudding in goat kids.

Hot-iron disbudding in goat kids causes acute pain and tissue damage. However, the duration of healing and wound sensitivity is unknown. We assessed wound healing and pain sensitivity in 18 female dairy goat kids disbudded with a heated iron at 10 d of age (range: 5-15 d). Pressure algometry was carried out twice a week from d 1 after disbudding to determine the mechanical nociceptive threshold (MNT) in 4 locations on each bud (front, lateral, caudal, and middle). At the same time, digital and infrared images of the wounds were used to visually and thermally describe the healing process. Wounds were visually scored daily for the presence of 7 tissue types: attached necrotic tissue, detached necrotic tissue, burns outside the necrotic ring, exudate, granulation, crust, and epithelium. All data were taken until epithelium was present for 4 consecutive days. Necrotic tissue detached completely from the scalp 26 ± 5 d after the procedure (mean ± SD; range: 17-43 d), and wounds took 50 ± 8 d (35-63 d) to re-epithelialize. Wounds were more sensitive at all stages of the healing process compared with re-epithelialized tissue. The caudal and middle locations were the most- and least-sensitive test sites (1.24 ± 0.10 and 1.90 ± 0.10 N, respectively; mean ± SE). Goats became less responsive to stimulation as their wounds decreased in size. Sensitivity did not differ between left and right sides of the head. Maximum surface temperature of necrotic tissue, when present, tended to be higher than that of epithelium (38.8 ± 0.2 vs. 38.1 ± 0.2°C); temperature did not differ among other tissue types. Our results indicate that hot-iron disbudding wounds in goats take, on average 7, wk to re-epithelialize (35-63 d) and are painful throughout this time, raising additional welfare concerns about this procedure.

Short communication: Changes under low ambient temperatures in the milk lipodome and metabolome of mid-lactation cows after dehorning as a calf.

Horns are living tissue and cows can use their horns for thermoregulatory purposes. We investigated the effect of the presence of horns on the metabolome of milk serum and lipidome of milk fat, to assess the physiological effect of dehorning. Milk sampling took place at low ambient temperatures of -6 to 2°C. Horned and dehorned cows were kept in a mixed herd of Holstein Friesian and Brown Swiss cows. The hypothesis was that horned cows needed to increase their metabolism to compensate for additional heat loss through the presence of their horns. No differences were observed in milk yield, milk solids, and somatic cell counts between horned and dehorned cows. For the milk metabolome, horned cows showed an upregulation of several glucogenic AA that could be transformed into glucose for energy supply and a downregulation of sugar intermediates and γ-glutamylcysteine compared with dehorned cows. The fatty acid (FA) composition in horned cows showed a shift toward decreased odd medium-chain FA (C7:0, C9:0, and C11:0) and increased cis-vaccenic acid (C18:1n-7 cis-11) and stearidonic acid (C18:4n-3). The changes in milk composition related to additional heat loss in horned cows indicate a competition in C3 metabolism for glucose synthesis and de novo FA synthesis under cold stress.

Iron and laterality effects on healing of cautery disbudding wounds in dairy calves.

Hot-iron disbudding, the process of cauterizing the horn buds of calves or goat kids at an early age to prevent horn growth, is routinely practiced in dairy production. The wounds take weeks to heal and are painful to touch throughout this time. Possible strategies to hasten the healing of disbudding wounds are not well understood, but the type of iron used may be an important factor to consider. When evaluating strategies to hasten healing, a within-subjects design may be preferable, but laterality effects might act as a potential source of variation and confounding in this type of experiment. Our objectives were to compare healing after disbudding with 2 commercially available irons, and to determine whether wounds healed differently on the left versus the right side of the head. Ten Holstein calves 4 to 10 d of age were disbudded using the Rhinehart X50A electric disbudder (Rhinehart Development Corp., Spencerville, IN) on one horn bud and the Portasol gas disbudder (Portasol USA, Elmira, OR) on the other; side (left vs. right) was balanced between treatments. We scored wounds daily for the presence of 6 tissue types: attached necrotic tissue, detached necrotic tissue, exudate, granulation, crust, and epithelium. Surface temperature and size of the wound were measured twice-weekly using thermal and digital photographs, respectively. The type of iron used did not affect latency to re-epithelialize, which took on average (mean ± standard deviation) 53 ± 3 d and 55 ± 3 d for Portasol and Rhinehart wounds, respectively (range: 40-70 d). However, compared with Portasol wounds, those from the Rhinehart had fewer days of granulation tissue and tended to have more days of detached necrotic tissue. The Portasol tip had a smaller total surface area than the Rhinehart, which may have resulted in a less severe burn, causing the necrotic tissue to fall off sooner. The left side tended to re-epithelialize faster than the right side (mean ± standard error: left 51 ± 3 d; right 57 ± 3 d) and have fewer days of crust. Left-sided wounds were also cooler and tended to be smaller than those on the right. To assess the external validity of these laterality effects in our primary experiment (experiment A), we analyzed wound healing data from 2 other disbudding studies, one on calves (experiment B) and one on goat kids (experiment C). We observed laterality effects in the opposite direction in Experiment B, but negligible effects in experiment C, indicating that the differences in laterality had low external validity; the biological meaning of this asymmetry is unclear. Nonetheless, if using a within-subjects design, asymmetries in wound healing should be considered to avoid confounding effects. In conclusion, wounds from both irons took 7 to 8 wk to heal, on average; other strategies to accelerate healing should be explored.

Structural adaptations to diverse fighting styles in sexually selected weapons.

The shapes of sexually selected weapons differ widely among species, but the drivers of this diversity remain poorly understood. Existing explanations suggest weapon shapes reflect structural adaptations to different fighting styles, yet explicit tests of this hypothesis are lacking. We constructed finite element models of the horns of different rhinoceros beetle species to test whether functional specializations for increased performance under species-specific fighting styles could have contributed to the diversification of weapon form. We find that horns are both stronger and stiffer in response to species-typical fighting loads and that they perform more poorly under atypical fighting loads, which suggests weapons are structurally adapted to meet the functional demands of fighting. Our research establishes a critical link between weapon form and function, revealing one way male-male competition can drive the diversification of animal weapons.

Deep homology and the origins of evolutionary novelty.

Do new anatomical structures arise de novo, or do they evolve from pre-existing structures? Advances in developmental genetics, palaeontology and evolutionary developmental biology have recently shed light on the origins of some of the structures that most intrigued Charles Darwin, including animal eyes, tetrapod limbs and giant beetle horns. In each case, structures arose by the modification of pre-existing genetic regulatory circuits established in early metazoans. The deep homology of generative processes and cell-type specification mechanisms in animal development has provided the foundation for the independent evolution of a great variety of structures.

Do pre- and post-copulatory sexually selected traits covary in large herbivores?

BACKGROUND: In most species, males compete to gain both matings (via pre-copulatory competition) and fertilizations (via post-copulatory competition) to maximize their reproductive success. However, the quantity of resources devoted to sexual traits is finite, and so males are predicted to balance their investment between pre- and post-copulatory expenditure depending on the expected pay-offs that should vary according to mating tactics. In Artiodactyla species, males can invest in weapons such as horns or antlers to increase their mating gains or in testes mass/sperm dimensions to increase their fertilization efficiency. Moreover, it has been suggested that in these species, males with territory defence mating tactic might preferentially increase their investment in post-copulatory traits to increase their fertilization efficiency whereas males with female defence mating tactic might increase their investment in pre-copulatory sexually selected traits to prevent other males from copulating with females. In this study, we thus test the prediction that male's weapon length (pre-copulatory trait) covaries negatively with relative testes size and/or sperm dimensions (post-copulatory traits) across Artiodactyla using a phylogenetically controlled framework. RESULTS: Surprisingly no association between weapon length and testes mass is found but a negative association between weapon length and sperm length is evidenced. In addition, neither pre- nor post-copulatory traits were found to be affected by male mating tactics. CONCLUSIONS: We propose several hypotheses that could explain why male ungulates may not balance their reproductive investment between pre- and post-copulatory traits.

Mechanical limits to maximum weapon size in a giant rhinoceros beetle.

The horns of giant rhinoceros beetles are a classic example of the elaborate morphologies that can result from sexual selection. Theory predicts that sexual traits will evolve to be increasingly exaggerated until survival costs balance the reproductive benefits of further trait elaboration. In Trypoxylus dichotomus, long horns confer a competitive advantage to males, yet previous studies have found that they do not incur survival costs. It is therefore unlikely that horn size is limited by the theoretical cost-benefit equilibrium. However, males sometimes fight vigorously enough to break their horns, so mechanical limits may set an upper bound on horn size. Here, I tested this mechanical limit hypothesis by measuring safety factors across the full range of horn sizes. Safety factors were calculated as the ratio between the force required to break a horn and the maximum force exerted on a horn during a typical fight. I found that safety factors decrease with increasing horn length, indicating that the risk of breakage is indeed highest for the longest horns. Structural failure of oversized horns may therefore oppose the continued exaggeration of horn length driven by male-male competition and set a mechanical limit on the maximum size of rhinoceros beetle horns.

Water footbath, automatic flushing, and disinfection to improve the health of bovine feet.

Disinfecting footbaths are used to treat and prevent interdigital dermatitis (ID) and heel horn erosion (HHE). However, many disinfectants are disadvantageous for the environment and, as an alternative, washing of the feet has been introduced. Our aim was to investigate the effect of water footbaths (trial 1), footbaths with CuSO4 (trial 2), automatic water flushing (trial 3), and water flushing followed by disinfection with a glutaraldehyde-based compound (trial 4) in 4 randomized controlled clinical trials performed in a freestall dairy herd of approximately 45 Norwegian Red cows. At trimming before and after each trial, hind foot diseases, hardness of the claw horn (in D-units), locomotion, and cleanliness of the claws were recorded. Before each trial, the cows were divided in comparable study and control groups, based on prevalence of ID and HHE, parity, and days in milk. Using a transponder-regulated gate, the study groups were led through a footbath (trials 1 and 2) or an automatic washer (trials 3 and 4), whereas the control groups were left untreated. Each trial lasted 3 mo and the curative effect in diseased cows and the preventive effect in cows with healthy feet on ID, HHE, and ID + HHE were analyzed. In trial 2, a preventive effect of CuSO4 on HHE compared with the untreated cows was observed. During trial 1, 100% (11/11) of the treated cows with ID got better and 22% (2/9) without ID became diseased, whereas 92% (11/12) of the treated cows with ID + HHE got better and 38% (3/8) without ID + HHE became diseased. During trial 2, 69% (9/13) of the treated cows with ID got better and 11% (1/9) without ID became diseased. During trial 4, 19% (3/16) of the untreated cows with ID + HHE got better and 71% (5/7) without ID + HHE became diseased. In trial 3, no significant effects on ID, HHE, or ID + HHE were revealed. In trial 2 (CuSO4), the treated cows' claw horn was harder after the trial compared with the controls (D-unit difference: 13.25). In trial 3 (stationary water flushing) the treated cows' claw horn was softer after the trial when compared with the controls (D-unit difference: -15.66). The CuSO4 footbaths were useful to prevent HHE and indicate that automatic stationary flushing with only water had no beneficial effect on ID or HHE. The claw horn of cows walking through CuSO4 became harder and the claw horn of cows that had their hind feet flushed with water became softer compared with the controls.

Evolution of horn length and lifting strength in the Japanese rhinoceros beetle Trypoxylus dichotomus.

What limits the size of nature's most extreme structures? For weapons like beetle horns, one possibility is a tradeoff associated with mechanical levers: as the output arm of the lever system-the beetle horn-gets longer, it also gets weaker. This "paradox of the weakening combatant" could offset reproductive advantages of additional increases in weapon size. However, in contemporary populations of most heavily weaponed species, males with the longest weapons also tend to be the strongest, presumably because selection drove the evolution of compensatory changes to these lever systems that ameliorated the force reductions of increased weapon size. Therefore, we test for biomechanical limits by reconstructing the stages of weapon evolution, exploring whether initial increases in weapon length first led to reductions in weapon force generation that were later ameliorated through the evolution of mechanisms of mechanical compensation. We describe phylogeographic relationships among populations of a rhinoceros beetle and show that the "pitchfork" shaped head horn likely increased in length independently in the northern and southern radiations of beetles. Both increases in horn length were associated with dramatic reductions to horn lifting strength-compelling evidence for the paradox of the weakening combatant-and these initial reductions to horn strength were later ameliorated in some populations through reductions to horn length or through increases in head height (the input arm for the horn lever system). Our results reveal an exciting geographic mosaic of weapon size, weapon force, and mechanical compensation, shedding light on larger questions pertaining to the evolution of extreme structures.

Data from selective harvests underestimate temporal trends in quantitative traits.

Human harvests can select against phenotypes favoured by natural selection, and natural resource managers should evaluate possible artificial selection on wild populations. Because the required genetic data are extremely difficult to gather, however, managers typically rely on harvested animals to document temporal trends. It is usually unknown whether these data are unbiased. We explore our ability to detect a decline in horn size of bighorn sheep (Ovis canadensis) by comparing harvested males with all males in a population where evolutionary changes owing to trophy hunting were previously reported. Hunting records underestimated the temporal decline, partly because of an increasing proportion of rams that could not be harvested because their horns were smaller than the threshold set by hunting regulations. If harvests are selective, temporal trends measured from harvest records will underestimate the magnitude of changes in wild populations.

Long horns protect Hestina japonica butterfly larvae from their natural enemies.

Animals sometimes have prominent projections on or near their heads serving diverse functions such as male combat, mate attraction, digging, capturing prey, sensing or defence against predators. Some butterfly larvae possess a pair of long frontal projections; however, the function of those projections is not well known. Hestina japonica butterfly larvae have a pair of long hard projections on their heads (i.e., horns). Here we hypothesized that they use these horns to protect themselves from natural enemies (i.e., predators and parasitoids). Field surveys revealed that the primary natural enemies of H. japonica larvae were Polistes wasps. Cage experiments revealed that larvae with horns intact and larvae with horns removed and fitted with horns of other individuals succeeded in defending themselves against attacks of Polistes wasps significantly more often than larvae with horns removed. We discuss that the horns counter the paper wasps' hunting strategy of first biting the larvae's 'necks' and note that horns evolved repeatedly only within the Nymphalidae in a phylogeny of the Lepidoptera. This is the first demonstration that arthropods use head projections for physical defence against predators.

Finite element analysis of ramming in Ovis canadensis.

The energy produced during the ramming of bighorn sheep (Ovis canadensis) would be expected to result in undesirable stresses in their frontal skull, which in turn would cause brain injury; yet, this animal seems to suffer no ill effects. In general, horn is made of an α-keratin sheath covering a bone. Despite volumes of data on the ramming behavior of Ovis canadensis, the extent to which structural components of horn and horn-associated structure or tissue absorb the impact energy generated by the ramming event is still unknown. This study investigates the hypothesis that there is a mechanical relationship present among the ramming event, the structural constituents of the horn, and the horn-associated structure. The three-dimensional complex structure of the bighorn sheep horn was successfully constructed and modeled using a computed tomography (CT) scan and finite element (FE) method, respectively. Three different three-dimensional quasi-static models, including a horn model with trabecular bone, a horn model with compact bone that instead of trabecular bone, and a horn model with trabecular bone as well as frontal sinuses, were studied. FE simulations were used to compare distributions of principal stress in the horn and the frontal sinuses and the strain energy under quasi-static loading conditions. It was noticed that strain energy due to elastic deformation of the complex structure of horn modeled with trabecular bone and with trabecular bone and frontal sinus was different. In addition, trabecular bone in the horn distributes the stresses over a larger volume, suggesting a mechanical link between the structural constituents and the ramming event. This phenomenon was elucidated through the principal stress distribution in the structure. This study will help designers in choosing appropriate material combinations for the successful design of protective structures against a similar impact.

Use of multivariate analysis as a tool in the morphological characterization of the main indigenous bovine ecotypes in northeastern Algeria.

Sustainability in livestock farming requires monitoring of autochthonous breeds which are well adapted to the local environment. The morphometric measurements seem to be the first approach which can provide useful information on the suitability of animal genetic resources for selection. In this work, thirteen morphometric variables were used for the phenotypic characterization of 130 adult autochthones cattle randomly selected from 30 local farms in Guelma. There were cases from four commonly accepted and traditional ecotypes: Guelmois, Cheurfa, Sétifien and Fawn. The results showed several and significant positive correlations between the different variables. Correlations were analyzed using Varimax orthogonal rotation PCA and three factors were extracted, which explain more than 75% of the total variation in the four ecotypes. Stepwise discriminant analysis showed that 6 of the 13 variables had discriminatory power to define the phenotypic profile of the ecotypes. Canonical discriminant analysis indicated that the Sétifien ecotype is separate from the other three ecotypes. Mahalanobis distances were significant between the different ecotypes except for the distance between the Guelmois and Fawn ecotypes. The cross-validation procedure assigned 91.42% of the Sétifien animals to their genetic group, while the percentages of animals assigned to the Cheurfa, Guelmois and Fawn ecotypes were 80.00%, 65.71% and 53.33% respectively. The multivariate approach has proven to be effective in differentiating the four ecotypes, with clear morphological differences from the Sétifien ecotype that may benefit from a genetic improvement program for more sustainable genetic resources preservation.

Reproductive competition promotes the evolution of female weaponry.

Secondary sexual traits in females are a relatively rare phenomenon. Empirical studies have focused on the role of male mate choice in their evolution; however, recently it has been suggested that secondary sexual traits in females are more likely to be under selection via reproductive competition. We investigated female competition and the influence of female phenotype on fitness in Onthophagus sagittarius, a species of dung beetle that exhibits female-specific horns. We compared reproductive fitness when females were breeding in competition versus breeding alone and found that competition for breeding resources reduced fitness for all females, but that smaller individuals suffered a greater fitness reduction than larger individuals. When females were matched for body size, those with the longest horns gained higher reproductive fitness. The fitness function was positive and linear, favouring increased horn expression. Thus, we present evidence that female body size and horn size in O. sagittarius are under directional selection via competition for reproductive resources. Our study is a rare example of female contest competition selecting for female weaponry.

Experimental study on the mechanical properties of the horn sheaths from cattle.

Bovine horn is composed of a sheath of keratin overlying a bony core. Previous studies of the bovine horn sheath have focused mainly on its morphology and compositions. In the present paper, we performed a series of uniaxial tension, three-point bending, and fracture tests to investigate the structural and mechanical properties of the horn sheaths from subadult cattle, Bos taurus. The effects of hydration on the mechanical properties were examined and their variations along the longitudinal direction of the horn sheath were addressed. Scanning electron microscopy of the fracture surfaces showed that the horn sheath has a layered structure and, more interestingly, the laminae have a rippled appearance. The Young's modulus and tensile strength increase from 850 MPa and 40 MPa at 19% water content to 2.3 GPa and 154 MPa at 0% water content, respectively. The Poisson's ratio of the horn sheath was about 0.38. The critical stress intensity factor was about 4.76 MPa m1/2 at an intermediate hydration (8% water content), greater than that at 0% water content (3.86 MPa m1/2) and 19% water content (2.56 MPa m1/2). The bending properties of the samples varied along the length of the horn. The mean flexural moduli of the specimens in the distal, middle and proximal parts were about 6.26 GPa, 5.93 GPa and 4.98 GPa, respectively; whereas the mean yield strength in the distal segment was about 152.4 MPa, distinctly higher than that in the middle (135.7 MPa) and proximal parts (116.4 MPa). This study deepens our understanding of the relationships among optimal structure, property and function of cattle horn sheaths.

Characterization of the poll allele in Brahman cattle using long-read Oxford Nanopore sequencing.

Brahman cattle (Bos indicus) are well adapted to thrive in tropical environments. Since their introduction to Australia in 1933, Brahman's ability to grow and reproduce on marginal lands has proven their value in the tropical beef industry. The poll phenotype, which describes the absence of horns, has become desirable in the cattle industry for animal welfare and handler safety concerns. The poll locus has been mapped to chromosome one. Four alleles, each a copy number variant, have been reported across this locus in B. indicus and Bos taurus. However, the causative mutation in Brahman cattle has not been fully characterized. Oxford Nanopore Technologies' minION sequencer was used to sequence four homozygous poll (PcPc), four homozygous horned (pp), and three heterozygous (Pcp) Brahmans to characterize the poll allele in Brahman cattle. A total of 98 Gb were sequenced and an average coverage of 3.33X was achieved. Read N50 scores ranged from 9.9 to 19 kb. Examination of the mapped reads across the poll locus revealed insertions approximately 200 bp in length in the poll animals that were absent in the horned animals. These results are consistent with the Celtic poll allele, a 212-bp duplication that replaces 10 bp. This provides direct evidence that the Celtic poll allele is segregating in the Australian Brahman population.

CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle.

In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein-Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.

Formation, structure, and function of extra-skeletal bones in mammals.

This review describes the formation, structure, and function of bony compartments in antlers, horns, ossicones, osteoderm and the os penis/os clitoris (collectively referred to herein as AHOOO structures) in extant mammals. AHOOOs are extra-skeletal bones that originate from subcutaneous (dermal) tissues in a wide variety of mammals, and this review elaborates on the co-development of the bone and skin in these structures. During foetal stages, primordial cells for the bony compartments arise in subcutaneous tissues. The epithelial-mesenchymal transition is assumed to play a key role in the differentiation of bone, cartilage, skin and other tissues in AHOOO structures. AHOOO ossification takes place after skeletal bone formation, and may depend on sexual maturity. Skin keratinization occurs in tandem with ossification and may be under the control of androgens. Both endochondral and intramembranous ossification participate in bony compartment formation. There is variation in gradients of density in different AHOOO structures. These gradients, which vary according to function and species, primarily reduce mechanical stress. Anchorage of AHOOOs to their surrounding tissues fortifies these structures and is accomplished by bone-bone fusion and Sharpey fibres. The presence of the integument is essential for the protection and function of the bony compartments. Three major functions can be attributed to AHOOOs: mechanical, visual, and thermoregulatory. This review provides the first extensive comparative description of the skeletal and integumentary systems of AHOOOs in a variety of mammals.