Progress and challenges in developing organoids in farm animal species for the study of reproduction and their applications to reproductive biotechnologies.

Within the past decades, major progress has been accomplished in isolating germ/stem/pluripotent cells, in refining culture medium and conditions and in establishing 3-dimensional culture systems, towards developing organoids for organs involved in reproduction in mice and to some extent in humans. Haploid male germ cells were generated in vitro from primordial germ cells. So were oocytes, with additional support from ovarian cells and subsequent follicle culture. Going on with the female reproductive tract, spherical oviduct organoids were obtained from adult stem/progenitor cells. Multicellular endometrial structures mimicking functional uterine glands were derived from endometrial cells. Trophoblastic stem cells were induced to form 3-dimensional syncytial-like structures and exhibited invasive properties, a crucial point for placentation. Finally, considering the embryo itself, pluripotent embryonic cells together with additional extra-embryonic cells, could self-organize into a blastoid, and eventually into a post-implantation-like embryo. Most of these accomplishments have yet to be reached in farm animals, but much effort is devoted towards this goal. Here, we review the progress and discuss the specific challenges of developing organoids for the study of reproductive biology in these species. We consider the use of such organoids in basic research to delineate the physiological mechanisms involved at each step of the reproductive process, or to understand how they are altered by environmental factors relevant to animal breeding. We evaluate their potential in reproduction of animals with a high genetic value, from a breeding point of view or in the context of preserving local breeds with limited headcounts.

Intestinal organoids in farm animals.

In livestock species, the monolayer of epithelial cells covering the digestive mucosa plays an essential role for nutrition and gut barrier function. However, research on farm animal intestinal epithelium has been hampered by the lack of appropriate in vitro models. Over the past decade, methods to culture livestock intestinal organoids have been developed in pig, bovine, rabbit, horse, sheep and chicken. Gut organoids from farm animals are obtained by seeding tissue-derived intestinal epithelial stem cells in a 3-dimensional culture environment reproducing in vitro the stem cell niche. These organoids can be generated rapidly within days and are formed by a monolayer of polarized epithelial cells containing the diverse differentiated epithelial progeny, recapitulating the original structure and function of the native epithelium. The phenotype of intestinal organoids is stable in long-term culture and reflects characteristics of the digestive segment of origin. Farm animal intestinal organoids can be amplified in vitro, cryopreserved and used for multiple experiments, allowing an efficient reduction of the use of live animals for experimentation. Most of the studies using livestock intestinal organoids were used to investigate host-microbe interactions at the epithelial surface, mainly focused on enteric infections with viruses, bacteria or parasites. Numerous other applications of farm animal intestinal organoids include studies on nutrient absorption, genome editing and bioactive compounds screening relevant for agricultural, veterinary and biomedical sciences. Further improvements of the methods used to culture intestinal organoids from farm animals are required to replicate more closely the intestinal tissue complexity, including the presence of non-epithelial cell types and of the gut microbiota. Harmonization of the methods used to culture livestock intestinal organoids will also be required to increase the reproducibility of the results obtained in these models. In this review, we summarize the methods used to generate and cryopreserve intestinal organoids in farm animals, present their phenotypes and discuss current and future applications of this innovative culture system of the digestive epithelium.

3D skin models in domestic animals.

The skin is a passive and active barrier which protects the body from the environment. Its health is essential for the accomplishment of this role. Since several decades, the skin has aroused a strong interest in various fields (for e.g. cell biology, medicine, toxicology, cosmetology, and pharmacology). In contrast to other organs, 3D models were mostly and directly elaborated in humans due to its architectural simplicity and easy accessibility. The development of these models benefited from the societal pressure to reduce animal experiments. In this review, we first describe human and mouse skin structure and the major differences with other mammals and birds. Next, we describe the different 3D human skin models and their main applications. Finally, we review the available models for domestic animals and discuss the current and potential applications.

Performance of Black Bengal goat: a 50-year review.

Black Bengal goat (BBG) is the most widely recognized legacy goat breed in Bangladesh. The breed is black in color yet likewise earthy, white, or dim colors additionally found. The breed has medium body size with grown-up weight 25-30 kg, little horns, short legs, and tight body structure. The BBG is one of the most compliant, all around adjusted, early maturing, prolific, productive, and tropical disease-resistant goat types of the world that produces incredible quality meat, milk, and skin. The breed is versatile in hot, moist, cruel, climatic conditions and flourishes well on a cacophonous dietary regimen from uncultivable decrepit grounds, residences, riversides, banks, sloping, and hilly territories where crop culture or dairy nourishing is inconceivable. In Bangladesh, the BBG is one of the main red meat-producing small ruminants which shares remarkable local interest during Eid-Ul-Adha, Eid-Ul-Fitr, wedding ceremony, birthday festival, circumcision, memorial programs, and other social celebrations with no social, cultural, and religious limitations. Being little in size, the BBG has been an amazing asset to advance supportable vocations for the negligible, little, and landless ranchers who rely upon free regular grazing lands for raising domesticated animals. Regardless of incredibly exceptional components and features, the production of BBG has not yet been popularized widely since meager consideration has been paid for improving their efficiency. Development of cutting edge hereditary, dietary, and health as well as disease control procedure and utilization of modern management frameworks may procure considerable changes in improving the overall performance of the BBG.

Genetic Variants Affecting Skeletal Morphology in Domestic Dogs.

Purebred dog breeds provide a powerful resource for the discovery of genetic variants affecting skeletal morphology. Domesticated and subsequently purebred dogs have undergone strong artificial selection for a broad range of skeletal variation, which include both the size and shapes of their bones. While the phenotypic variation between breeds is high, within-breed morphological variation is typically low. Approaches for defining genetic variants associated with canine morphology include quantitative within-breed analyses, as well as across-breed analyses, using breed standards as proxies for individual measurements. The ability to identify variants across the genomes of individual dogs can now be paired with precise measures of morphological variation to define the genetic interactions and the phenotypic effect of variants on skeletal morphology.

Identification of working reindeer using palaeopathology and entheseal changes.

OBJECTIVE: This paper explores the potential of analyzing pathological lesions and entheseal changes in the identification of working reindeer. METHODS: The skeletons of 26 modern working reindeer from Siberia and Northern Finland are analyzed for pathological lesions and entheseal changes. RESULTS: Working results in elevated frequencies of pathological lesions, specifically joint disease in cervical and thoracic vertebrae, humeri, os coxae and proximal phalanges. Entheseal scores indicate the intensified use of shoulder flexors and extensors, and possibly elbow, hip and knee flexors and extensors in working reindeer. CONCLUSION: Patterns of skeletal changes can be used in the identification of working reindeer from the past. SIGNIFICANCE: This study provides first evidence that pathological lesions and entheseal changes can be used to assess draught and cargo use of reindeer. Therefore, the methods presented in this study provide an opportunity to scrutinize past reindeer herding practices, reindeer domestication and human-reindeer cooperation. LIMITATIONS: Methods need to be applied with caution due to the multifactorial etiologies of pathological lesions and entheseal changes. SUGGESTIONS FOR FUTURE RESEARCH: We suggest that in future studies, these methods are applied to archaeological material accompanied by osteometric and contextual analyses.

Earliest evidence for equid bit wear in the ancient Near East: The "ass" from Early Bronze Age Tell es-Sâfi/Gath, Israel.

Analysis of a sacrificed and interred domestic donkey from an Early Bronze Age (EB) IIIB (c. 2800-2600 BCE) domestic residential neighborhood at Tell es-Sâfi/Gath, Israel, indicate the presence of bit wear on the Lower Premolar 2 (LPM2). This is the earliest evidence for the use of a bit among early domestic equids, and in particular donkeys, in the Near East. The mesial enamel surfaces on both the right and left LPM2 of the particular donkey in question are slightly worn in a fashion that suggests that a dental bit (metal, bone, wood, etc.) was used to control the animal. Given the secure chronological context of the burial (beneath the floor of an EB IIIB house), it is suggested that this animal provides the earliest evidence for the use of a bit on an early domestic equid from the Near East.

The evolution of ontogenetic allometric trajectories in mammalian domestication.

Morphological divergence of domesticated as compared to wild forms must result from changes in the ontogenetic process. Species-specific tests for heterochrony have rejected a single explanation of domestic forms representing juveniles of their wild relatives. Ontogenetic allometric trajectories for 12 pairs of wild and domestic mammals were examined using skull growth data for 1070 specimens, including representatives from all lineages in which domestication has occurred. A suite of tests were performed to quantify allometric disparity in wild and domestic forms and assess the extent and patterning of modification to allometric trajectories. Domestication has modified postnatal ontogenetic allometric trajectories in mammals, and has generated disparity, achieved through lengthening of trajectory slopes and alteration to slope angles. Allometric disparity was similar for domestic forms compared to their wild relatives, whereas the magnitude of dispersion along allometric vectors differed between precocial mammals and altricial mammals, underscoring the importance of life history and shared evolutionary history in patterns of ontogenetic variation. The results verify the importance of scaling in the morphological changes associated with domestication. The response to domestication for all measured trajectory parameters was variable across species, suggesting multiple pathways of change.

Production of accurate skeletal models of domestic animals using three-dimensional scanning and printing technology.

Access to adequate anatomical specimens can be an important aspect in learning the anatomy of domestic animals. In this study, the authors utilized a structured light scanner and fused deposition modeling (FDM) printer to produce highly accurate animal skeletal models. First, various components of the bovine skeleton, including the femur, the fifth rib, and the sixth cervical (C6) vertebra were used to produce digital models. These were then used to produce 1:1 scale physical models with the FDM printer. The anatomical features of the digital models and three-dimensional (3D) printed models were then compared with those of the original skeletal specimens. The results of this study demonstrated that both digital and physical scale models of animal skeletal components could be rapidly produced using 3D printing technology. In terms of accuracy between models and original specimens, the standard deviations of the femur and the fifth rib measurements were 0.0351 and 0.0572, respectively. All of the features except the nutrient foramina on the original bone specimens could be identified in the digital and 3D printed models. Moreover, the 3D printed models could serve as a viable alternative to original bone specimens when used in anatomy education, as determined from student surveys. This study demonstrated an important example of reproducing bone models to be used in anatomy education and veterinary clinical training. Anat Sci Educ 11: 73-80. © 2017 American Association of Anatomists.

Morphometry of the coronary ostia and the structure of coronary arteries in the shorthair domestic cat.

The aim of this study was to measure the area of the coronary ostia, assess their localization in the coronary sinuses and to determine the morphology of the stem of the left and right coronary arteries in the domestic shorthair cat. The study was conducted on 100 hearts of domestic shorthair cats of both sexes, aged 2-18 years, with an average body weight of 4.05 kg. A morphometric analysis of the coronary ostia was carried out on 52 hearts. The remaining 48 hearts were injected with a casting material in order to carry out a morphological assessment of the left and right coronary arteries. In all the studied animals, the surface of the left coronary artery ostium was larger than the surface of the right coronary artery ostium. There were four types of the left main coronary artery: type I (23 animals, 49%)-double-branched left main stem (giving off the left circumflex branch and the interventricular paraconal branch, which in turn gave off the septal branch), type II (12 animals, 26%)-double-branched left main stem (giving off the left circumflex branch and the interventricular paraconal branch without the septal branch), type III (11 animals, 23%)-triple-branched left main stem (giving off the left circumflex branch, interventricular branch and the septal branch, type IV (1 animal, 2%)-double-branched left main stem (giving off the interventricular paraconal branch and the left circumflex branch, which in turn gave off the septal branch). The left coronary artery ostium is greater than the right one. There is considerable diversity in the branches of proximal segment of the left coronary artery, while the right coronary artery is more conservative. These results can be useful in defining the optimal strategies in the endovascular procedures involving the coronary arteries or the aortic valve in the domestic shorthair cat.

Spread of domestic animals across Neolithic western Anatolia: New zooarchaeological evidence from Ugurlu Höyük, the island of Gökçeada, Turkey.

The zooarchaeological research presented here investigates Neolithic and Chalcolithic (ca. 6500-5000 cal. BC) animal exploitation strategies at Ugurlu Höyük on the Turkish island of Gökçeada in the northeastern Aegean Sea. Toward this end, we first discuss the results of our analysis of the zooarchaeological assemblages from Ugurlu Höyük and then consider the data within a wider regional explanatory framework using a diachronic approach, comparing them with those from western and northwestern Anatolian sites. The first settlers of Gökçeada were farmers who introduced domestic sheep, goats, cattle and pigs to the island as early as 6500 years BC. Our results align well with recently published zooarchaeological data on the westward spread of domestic animals across Turkey and the Neolithization of southeast Europe. Using an island site as a case study, we independently confirm that the dispersal of early farming was a polynucleated and multidirectional phenomenon that did not sweep across the land, replace everything on its way, and deliver the same "Neolithic package" everywhere. Instead, this complex process generated a diversity of human-animal interactions. Thus, studying the dispersal of early farmers from southwest Asia into southeast Europe via Anatolia requires a rigorous methodological approach to develop a fine-resolution picture of the variability seen in human adaptations and dispersals within complex and rapidly changing environmental and cultural settings. For this, the whole spectrum of human-animal interactions must be fully documented for each sub-region of southwest Asia and the circum-Mediterranean.

Morphometric Wings Similarity among Sylvatic and Domestic Populations of Triatoma infestans (Hemiptera: Reduviidae) from the Gran Chaco Region of Paraguay.

Despite sustained efforts for eliminating Triatoma infestans, reinfestation still persists in large part of the endemic area of Chagas disease from the Gran Chaco region. Sylvatic T. infestans populations seem to threat success of control programs of domestic T. infestans. In this study, we analyze whether T. infestans collected after a community-wide spraying were survivors or were immigrants from elsewhere using geometric morphometric tools. We used 101 right wings of female T. infestans captured before and after intervention program carried out in 12 de Junio and Casuarina, villages from Paraguayan Chaco, and in Puerto Casado during presprayed collection. There were no significant differences in wing size of domestic T. infestans between pre- and postspraying populations, and between domestic and sylvatic ones. When shape variables originating from postintervention individuals from 12 de Junio were introduced one by one into a discriminant analysis, the greatest weight (53%) was allocated to the sylvatic group. Furthermore, from the prespraying population, 25% were reallocated as postintervention individuals. Only 11% of the insects were reassigned to other groups Puerto Casado and Casuarina. These results suggest that postspraying individuals appear to have different origins. Half of the postspraying individuals from 12 de Junio were similar to the sylvatic ones and 25% of these were similar to those captured in the prespraying period. This remarkable morphometric wings similarity between sylvatic and domestic populations is new evidence suggesting that they could be highly related to each other in the Paraguayan Chaco; human-fed bugs from sylvatic area also support this.

Brain size is reduced by selection for tameness in Red Junglefowl- correlated effects in vital organs.

During domestication animals have undergone changes in size of brain and other vital organs. We hypothesize that this could be a correlated effect to increased tameness. Red Junglefowl (ancestors of domestic chickens) were selected for divergent levels of fear of humans for five generations. The parental (P0) and the fifth selected generation (S5) were culled when 48-54 weeks old and the brains were weighed before being divided into telencephalon, cerebellum, mid brain and optic lobes. Each single brain part as well as the liver, spleen, heart and testicles were also weighed. Brains of S5 birds with high fear scores (S5 high) were heavier both in absolute terms and when corrected for body weight. The relative weight of telencephalon (% of brain weight) was significantly higher in S5 high and relative weight of cerebellum was lower. Heart, liver, testes and spleen were all relatively heavier (% of body weight) in S5 high. Hence, selection for tameness has changed the size of the brain and other vital organs in this population and may have driven the domesticated phenotype as a correlated response.

Associations between Domestic-Dog Morphology and Behaviour Scores in the Dog Mentality Assessment.

The domestic dog shows a wide range of morphologies, that humans have selected for in the process of creating unique breeds. Recent studies have revealed correlations between changes in morphology and behaviour as reported by owners. For example, as height and weight decrease, many undesirable behaviours (non-social fear, hyperactivity and attention seeking) become more apparent. The current study aimed to explore more of these correlations, but this time used reports from trained observers. Phenotypic measurements were recorded from a range of common dog breeds (n = 45) and included cephalic index (CI: the ratio of skull width to skull length), bodyweight, height and sex. These data were then correlated with results from the Dog Mentality Assessment (DMA), which involves trained observers scoring a dog's reaction to stimuli presented over 10 standardised subtests. Each subtest is designed to evoke a behavioural response. Backward elimination and weighted step-wise regression revealed that shorter dogs demonstrated more aggressive tendencies, reacting defensively toward both assistants dressed as ghosts (p = 0.045), and to a dummy (p = 0.008). Taller dogs were more affectionate when greeting and being handled by humans (p = 0.007, p = <0.001, respectively). Taller dogs were also more cooperative (p = <0.001), and playful (p = 0.001) with humans than shorter dogs. Heavier dogs were more inquisitive toward a dummy (p = 0.011), to the source of a metallic noise (p = 0.010) and to an assistant (p = 0.003). Heavier dogs were also more attentive to the ghosts (p = 0.013). In comparison, lighter dogs were cautious of a dummy (p = <0.001) and fearful of the sound of a gunshot (p = <0.001). Lighter dogs were also cautious of, and demonstrated prolonged fearfulness toward, the source of metallic noise (p = <0.001, p = <0.034, respectively). With a far larger sample and the advantage of third-party reporting (which overcomes potential owner bias), the current findings build on previous studies in this field, further supporting covariance between morphology and behaviour.

Comparative morphological evaluation of domestic animal cornea.

OBJECTIVE: This study described corneal morphology in different domestic animals using histological and immunohistochemical methods. Specifically, we evaluated the species-specific distribution pattern of cytokeratins (CKs) and aquaporins (AQPs) to assess their contribution to the strength and transparency of the cornea, respectively. PROCEDURES: Corneal sections (2 µm thick) were obtained from 28 pigs, 11 cows, two goats, six horses, four dogs, and five cats and stained with hematoxylin-eosin and periodic acid-Schiff (PAS) reaction. Immunohistochemistry was also performed using CK1 to 3 and AQP1 and 5 antibodies. RESULTS: Domestic animal corneas were composed of corneal epithelium, stroma, Descemet's membrane, and corneal endothelium. Bowman's layer was not detected using the PAS reaction. The three-layered epithelium was thinnest in carnivores and thickest in equines and bovines. CKs were demonstrated immunohistochemically in all species examined, especially in the most superficial layer of the corneal epithelium. CKs were more widely distributed in canine and feline corneal epithelial cell layers compared to other species. The corneal monolayer endothelium was immunostained with AQP1 in all species examined, and it was also present in stromal keratocytes in a species-specific manner. In contrast, AQP5 was exclusively localized to the corneal epithelium. Epithelial staining patterns varied markedly between species, and the widest distribution of AQP5 was demonstrated in feline epithelial cell layers. CONCLUSIONS: Differences in the distribution of CKs and AQPs in various species suggest species differences in the maintenance of structural integrity and fluid balance.

Rabbit genome analysis reveals a polygenic basis for phenotypic change during domestication.

The genetic changes underlying the initial steps of animal domestication are still poorly understood. We generated a high-quality reference genome for the rabbit and compared it to resequencing data from populations of wild and domestic rabbits. We identified more than 100 selective sweeps specific to domestic rabbits but only a relatively small number of fixed (or nearly fixed) single-nucleotide polymorphisms (SNPs) for derived alleles. SNPs with marked allele frequency differences between wild and domestic rabbits were enriched for conserved noncoding sites. Enrichment analyses suggest that genes affecting brain and neuronal development have often been targeted during domestication. We propose that because of a truly complex genetic background, tame behavior in rabbits and other domestic animals evolved by shifts in allele frequencies at many loci, rather than by critical changes at only a few domestication loci.

Keratinization of the esophageal epithelium of domesticated mammals.

We studied the esophageal epithelium for keratinization characteristics from samples of domesticated mammals of three nutrition groups (herbivores: horse, cattle, sheep; omnivores: pig, dog, rat; carnivores: cat) using histochemistry (keratins, disulfides), sulfur measurements, and cryo-SEM. Keratins were found in all esophageal layers of all species, except for the equine Stratum corneum. The positive reaction staining of Pan-keratin was remarkable, but decreased in intensity toward the outer layers, whereas in the pig and cat, staining was confined to the corneal layer. The herbivores revealed positive staining reactions in the upper Stratum spinosum, particularly in the sheep. Regarding single keratins, CK6 immunostating was found in most esophageal layers, but only weakly or negatively in the porcine and equine Stratum corneum. CK13 staining was restricted to the sheep and here was found in all layers. CK14 could be detected in the equine and feline Stratum basale, and upper vital layers of the dog and rat. CK17 appeared only in the Stratum spinosum and Stratum granulosum, but in all layers of the dog and cat. Disulfides reacted strongest in the Stratum corneum of the herbivores, as corroborated by the sulfur concentrations in the esophagus. Our study emphasized that keratins are very important for the mechanical stability of the epithelial cells and cell layers of the mammalian esophagus. The role of these keratins in the esophageal epithelia is of specific interest owing to the varying feed qualities and mechanical loads of different nutrition groups, which have to be countered.