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Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.

Adult T‐cell leukemia (ATL) is a mature T‐cell malignancy caused by human T‐cell leukemia virus type I infection, and 10%‐25% of patients show central nervous system (CNS) involvement. CNS involvement significantly reduces survival and there are no effective treatments for CNS involvement. Therefore, an appropriate animal model is required to evaluate the inhibitory effects of novel drugs on the progression of ATL with CNS involvement. Here, we established a mouse model of ATL with CNS involvement using NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ mice inoculated with ATL cells intramuscularly in the postauricular region, and these mice showed paraparesis. Of the 10 mice inoculated with ATL cells intramuscularly (I.M.) at 5 weeks of age, 8 (80%) showed paraparesis, whereas none of the 10 mice inoculated with ATL cells subcutaneously (S.C.) showed paraparesis. In the I.M. group, PCR detected HTLV‐1‐specific genes in the thoracic and lumbar vertebrae; however, in the S.C. group, the vertebrae were negative for HTLV‐1 genes. Histological analysis revealed a particularly high incidence of tumors, characterized by accumulation of the injected cells, in the thoracic vertebrae of mice in the I.M. group. Tumor cell infiltration was relatively high in the bone marrow. Spinal cord compression caused by invasion of the tumor mass outside the pia mater was observed in the thoracic vertebrae of the spinal cord. In conclusion, we have reported a mouse model of tumor growth with paraparesis that may be used to assess novel therapeutic agents for ATL with CNS involvement.

This study demonstrated the potential of using urea and urea fertilizer to neutralize formaldehyde (Fd) in chicken cadavers. Initially, in vitro Fd neutralization with various concentrations of urea solution (US) and urea fertilizer solution (UFS) was conducted; subsequently, 18% US and 27% UFS were selected for infusing into the formalinized chickens. The measurement at 48 hr after infusion showed that both solutions could effectively lower Fd in chicken cadavers to below a permissible exposure limit without affecting cadaveric and histological quality. In addition, neutralizing power of 18% US was approximately 1.3 times that of 27% UFS. This is the first demonstration of neutralizing potential of US and UFS against Fd both in vitro and in vivo.

Ehlers-Danlos syndrome (EDS) is a group of hereditary diseases caused by mutation of extracellular matrix-related genes. Recently, spondylodysplastic EDS-Zip13 (spEDS-Zip13: OMIM 612350) was recognized as a new EDS type. This current study could reveal various morphometric differences of collagenous population in the proper substance of cornea between the wild type and spEDS-Zip13-knockout (Zip13-KO) mice. Blockade of Smad-signaling pathway might initiate these alterations. Predilected dissimilarity in level of transcriptional activity probably dictated morphology of keratocyte and shape and electron density of its nucleus. In addition, the imbalance of proteoglycans and glycosaminoglycans would also affect the diameter and arrangement of collagen fibrils. These findings would be considered as vulnerable characteristics of corneal stroma of the Zip13-KO mice.

Superficial digital flexor tendon (SDFT) of the bovine hindlimb originates from the caudolateral aspect of the distal femur and finally inserts onto the plantar aspect of the middle phalanges. In the present study, morphology and morphometry of the bovine SDFT at the muscle-tendon junction (MTJ), middle metatarsus (mM) and tendon-bone interface (TBI) were investigated. Cross-sectional morphology at the three regions of SDFT were oval, semioval and ring-formed, respectively. Significant difference in cross-sectional area was found only between MTJ-mM and mM-TBI (P

Ingestion of collagen peptide (CP) elicits beneficial effects on the body, including improvement in blood lipid profiles, but the underlying mechanisms remain unclear. The purpose of this study was to investigate the effects of CP ingestion on the liver, which controls lipid metabolism in the body. Male BALB/cCrSlc mice were bred with the AIN-93M diet containing 14 % casein or the AIN-93M-based low-protein diet containing 10 % casein or a diet containing 6 % casein+4 % CP for 10 weeks (n 12/group). Total, free and esterified cholesterol levels in the blood decreased in the CP group. DNA microarray analysis of the liver revealed that expressions of genes related to lipid metabolic processes such as the PPAR signalling pathway and fatty acid metabolism increased in the CP group compared with the 10 % casein group. The expressions of several genes involved in steroid metabolic process, including Cyp7a1 and Cyp8b1, were decreased, despite being targets of transcriptional regulation by PPAR. These data suggest that lipid metabolism in the liver is altered by CP ingestion, and the decrease in blood cholesterol levels in the CP group is not due to enhancement of the steroid metabolic process. On the other hand, expressions of genes related to the unfolded protein response (UPR) significantly decreased at the mRNA level, suggesting that CP ingestion lowers endoplasmic reticulum stress. Indeed, protein levels of phosphorylated inositol-requiring enzyme 1 decreased after CP ingestion. Taken together, CP affects the broader pathways in the liver – not only lipid metabolism but also UPR.

The fine structures of different tendons in various animals at different ages have been studied extensively to reveal their arrangement and growth patterns. However, knowledge of the microstructures of the growing tenocytes in the tendons of piglets is still lacking. Thus, we performed the first morphometric analysis to describe the characteristics of tenocytes in the metacarpal superficial digital flexor tendon of 0-, 10- and 20-day-old piglets. In the present study, hydrochloric acid/collagenase digestion was applied to remove the interstitial connective tissue to obtain clear visualization of intact tenocytes and their cytoplasmic processes (Cp). Then, the morphometry of the tenocytes was investigated by optical and electron microscopy. The mean ± SE values of the fascicle area, number of tenocytes/fascicle, cell density, number of Cp/tenocyte, length of Cp, and thickness of Cp were compared among the three age groups. Significant differences (judged at P

In horse, the characterizations of hyalocytes under the steady state are still unclear. Therefore, we investigated characterizations of hyalocytes in normal equine eyes by their immunohistochemical phenotype, histomorphology and distribution. Thirty-one eyes from 18 horses, divided into 4 groups (G) by age, were used: early (G1) and late gestation (G2) fetuses, 1- to 3-year-old (G3) and 8- to 24-year-old (G4) horses. Equine hyalocytes were histologically classified into 4 types, and they immunohistochemically expressed MHC II and CD163. Hyalocytes were detected on and/or around ciliary processes and pars plana in G2, G3 and G4, but were not located on retina and optic papilla. A significant increase in distribution was found between G2 and both G3 and G4, and the largest distribution was found at ciliary processes in these groups. Equine hyalocytes were characterized as residential ocular macrophage and MHC II antigen-bearing cell, accompanied by a pleomorphic appearance and located in the contiguous ciliary body. Our data provided characterizations of hyalocytes in normal equine eyes and may well contribute to improving the understanding of pathogenesis of equine ocular disease.

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-β1 (TGF-β1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-β1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-β1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-β1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-β1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-β homeostasis.

KASEA16(+) and KASEA16(−) peptides, the net charges of which are positive and negative, respectively, under a neutral condition could undergo self-assembly into nanofibers and form transparent hydrogels without peptide aggregation upon rapid pH neutralization. The numbers of NIH3T3 cells attached to the KASEA16(+) hydrogel and KASEA16(−) hydrogel were similar, and cells proliferated with time on both hydrogels. Cells on the KASEA16(+) hydrogel had spindle-like morphology, while cells on the KASEA16(−) hydrogel formed clusters without extending cytoplasmic processes. Comparison of differently charged peptides under a neutral condition suggested that the charges of the scaffolds should be taken into consideration for the best design and selection of scaffolds for cell culture. Since the KASEA16(+) peptide could form a stable hydrogel under a neutral condition and the hydrogel served as a scaffold for cell proliferation, the KASEA16(+) hydrogel will be a useful scaffold for cell culture.

Beneficial effects of collagen peptide ingestion to reduce serum triacylglycerides have been reported, suggesting that the physiological conditions of adipocytes are modulated following collagen peptide inges- tion. In this study, the effects of prolylhydroxyproline, a major digestive product of ingested collagen pep- tide in the blood, on the differentiation of mouse 3T3-L1 preadipocytes in vitro were investigated. 3T3-L1 preadipocytes were induced to differentiate to adipocytes and treated with prolylhydroxyproline, or with an amino acid mixture of proline and hydroxyproline as a control. The amount of lipid was not affected by these treatments. However, the size of the lipid droplet was significantly smaller when treated with prolylhydroxyproline compared to the amino acid mixture or the non-treated control. Proton-coupled oligopeptide transporters were expressed in non-differentiated and/or differentiated 3T3-L1 cells. These results suggest that prolylhydroxyproline might modulate the morphology of lipid droplets by incorpora- tion into adipocytes through the transporters.

J Oral Pathol Med (2010) 39: 617–623 Objective: To examine the effects of polaprezinc on morphologic change of the tongue epithelium and on cell cycle regulation of taste bud cells by using zinc-deficient rats, an animal model of taste disturbance. Methods: After 28 days of feeding with zinc-sufficient or -deficient diet, the rats fed a zinc-deficient diet were divided into four groups in which 0, 1, 3 and 10 mg/kg of polaprezinc were administered for 28 days with continuation of diet. Histopathological and morphological examinations of the tongue were carried out. Results: Parakeratosis was observed in all rats receiving the zinc-deficient diet and 1 mg/kg polaprezinc but not in rats receiving 3 and 10 mg/kg polaprezinc. The ratio of keratinizing epithelium in the outer and inner circumference were significantly increased from 9.6% and 11.3%, respectively, in zinc-sufficient rats to 36.9% and 32.9%, respectively, in zinc-deficient rats (P

The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo . The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were largest in the SDFT and smallest in the CDET. Addition of TNFα to culture of all three types of tendinocytes increased the synthesis of both proMMP-9 (except CDET) and collagen and decreased proMMP-13 synthesis and had no effect on proMMP-2 synthesis. Flexor tendons in forelimbs (SDFT and DDFT) restore energy during locomotion and are more easily injured than are extensor tendons. This structural property would cause active ECM and MMPs synthesis. And CDET have very low turnover potential; in the small number of cells, low cellular proliferation, lower ability for synthesis of collagen and MMPs. The isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and responsiveness of tendinocytes to complex inflammatory responses in tendinopathy.

Epithelioid cells are found in lymph nodes in cases of metastatic carcinoma; however, epithelioid cells with benign features have also been discovered incidentally in lymph nodes. Epithelioid cells were observed in mediastinal lymph nodes of cattle without cancer during routine diagnostic necropsy. To explain this finding, the authors evaluated the prevalence and histopathologic, immunohistochemical, and ultrastructural features retrospectively using mediastinal lymph nodes from 110 cattle and found that 66 of the lymph nodes contained epithelioid cells. In all 66 nodes, most of the epithelioid cells were individual or aggregated in nodal sinuses; in 21 nodes, some epithelioid cells formed tubular structures. The individual and aggregated epithelioid cells were mostly considered to be mesothelial in origin by immunohistochemical and ultrastructural examination; presumably, they entered the lymph nodes via lymphatic vessels, because they were found in afferent lymphatic vessels and nodal sinuses. Although the presence of epithelioid cells in lymph nodes prompts suspicion of metastatic carcinoma, the epithelioid cells in these bovine lymph nodes did not disrupt nodal architecture, lacked atypia or mitotic figures, and did not invade nodal parenchyma.

Purpose: To evaluate the effectiveness of atelocollagen for canine corneal wound healing. Materials and Methods: Atelocollagen was used to fill a transplant bed in the central cornea, which was then covered with a contact lens. The wound healing process was analyzed clinically, morphologically, and biochemically. Results: At the early healing stage, both the pupillary zone and details of the iris were observed. The stromal collagen fibrils normalized in a time-dependent manner. Type III collagen in the wound area was detected faintly throughout the experimental period. Conclusions: This novel method is advantageous for accelerating wound healing without causing inflammation.

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-β1 (TGF-β1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-β1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-β1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-β1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-β1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-β homeostasis.

Myometrial strips isolated from non-pregnant pigs show spontaneous contractile activity. In the present study, the involvement of endogenous prostaglandins in regulation of uterine spontaneous contraction was investigated using mechanical, immunohistochemical and biochemical approaches. Immunohistochemical study and Western blot analysis for immunoreactive cyclooxygenase (COX) indicated that COX-1 but not COX-2 was expressed predominantly in the myometrium of non-pregnant pigs in a muscle layer-dependent manner (longitudinal muscle > circular muscle). Pretreatment of uterine strips with indomethacin and selective COX-1 inhibitors (SC-560 and FR122047) significantly reduced both the amplitude and frequency of spontaneous contraction in the longitudinal muscle, but inhibition by COX inhibitors was negligible in the circular muscle. On the other hand, CAY10404, a COX-2 inhibitor, did not change the spontaneous contraction in either of the muscle layers. Pretreatment with SC-560 reduced myometrial PGF2α and PGE2 levels. Contractile FP and EP3 receptors were expressed in a muscle layer-dependent manner (longitudinal muscle > circular muscle), similar to the expression pattern of COX-1. In conclusion, endogenous prostaglandins produced by COX-1 regulate spontaneous contractile activity of non-pregnant porcine uterine longitudinal muscle selectively due to the heterogeneous expression of contractile prostanoid receptors and COX-1.

The fine structure in the center and periphery of the cornea of 16 beagle dogs were characterized and compared. The central cornea (about 540 microm) was apparently thinner than the peripheral cornea (about 720 microm). Thickness ratios of the corneal substantia propria to the entire cornea were approximately 86% in both portions. In addition, number of collagen lamellae, collagen fibril diameter, and collagen fibril index of the central substantia propria are different from those of the periphery (253 vs 236 lamellae, 29.1 vs 32.0 nm, and 39.0 vs 41.6%, respectively). These differences are thought to be due to site-dependent accumulation of proteoglycans (decorin and lumican) which are responsible for production of thin fibrils. The central portion with higher proteoglycans would have abundant thin fibrils with less slippage but better elasticity to buffer against the direct impact of intraocular pressure on the cornea. In contrast, thick fibrils in the peripheral substantia propria would contribute to the maintenance of tensile strength acting on the transition zone between the cornea and sclera.

A defect of the core protein of dermatan sulfate proteoglycan was suspected in a Holstein calf affected with a variant form of Ehlers-Danlos syndrome. The mutation was a guanine-to-adenine transition at nucleotide position 254, which resulted in a serine-to-asparagine substitution of the bovine proteoglycan core protein. This substitution occurred in the serine-glycine dipeptide repeat that was suspected to be the binding portion of dermatan sulfate. This point mutation in the genome was also detected by the use of restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) methods. The results of the RFLP and PCR indicated that the calf was a heterozygote of an abnormal gene and a normal gene of the core protein. The interpretation of these data revealed that the functional abnormality in cutaneous tissues of the calf was caused by an abnormal gene of the proteoglycan core protein, which induced a substitution of amino acid.

The ultrastructure of cardiac muscle from 3 aged one-humped camels (Camelus dromedarius) was examined to see if aged camels also develop hypertrophic Z-lines as observed in the heart of aged humans and other animals. The results proved that the aged camel heart develops hypertrophic Z-lines together with other age-associated alterations, including loss of myofibrils and myofilaments, increase of intermyofibrillar spaces and intercellular spaces, and disruption of mitochondrial cristae and intercalated disc structure. Hypertrophic Z-lines are somewhat smaller than those of other species, but occur both in the atria and ventricles. Although the majority of cardiac muscle samples revealed a normal structure, the finding of hypertrophic Z-lines in camel heart is the first such observation in a ruminant animal. The anomalous camel muscle resembles that of other species in terms of cell alterations commonly associated with aged hearts.

To examine the effects of polaprezinc on morphologic change of the tongue epithelium and on cell cycle regulation of taste bud cells by using zinc-deficient rats, an animal model of taste disturbance.After 28 days of feeding with zinc-sufficient or -deficient diet, the rats fed a zinc-deficient diet were divided into four groups in which 0, 1, 3 and 10 mg/kg of polaprezinc were administered for 28 days with continuation of diet. Histopathological and morphological examinations of the tongue were carried out.Parakeratosis was observed in all rats receiving the zinc-deficient diet and 1 mg/kg polaprezinc but not in rats receiving 3 and 10 mg/kg polaprezinc. The ratio of keratinizing epithelium in the outer and inner circumference were significantly increased from 9.6% and 11.3%, respectively, in zinc-sufficient rats to 36.9% and 32.9%, respectively, in zinc-deficient rats (P0.001 and0.01). This increase was reversed to 13.7% and 12.3% in rats that received 3 and 10 mg/kg polaprezinc in the outer circumference, respectively. Same phenomenon was seen in the inner circumference part, 13.0% and 10.8% (P0.01), respectively. In addition, proliferating cell nuclear antigen-positive cells in the taste bud were significantly decreased from 75.5% in zinc-sufficient rats to 32.2% in zinc-deficient rats (P0.001). This decrease was reversed to 70.3%, 83.1% and 81.2% in rats that received 1, 3 and 10 mg/kg polaprezinc, respectively.Polaprezinc improves parakeratosis and decreases taste bud cell proliferation caused by zinc deficiency. These effects may be involved in mechanisms underlying improvement of taste disorders in animal models.

The anatomical features of congenital duplication in the bovine calf encountered in 39 cases over eleven years in Hokkaido were investigated macroscopically. Among the animals studied, 14 were male, 20 female and 5 of unknown gender, and the anomaly was noted in 35 Holstein-Friesians, 3 Japanese Blacks and 1 Hereford. The duplications observed in this study were classified by four types: free asymmetrical, 4 (all 4 acardius); attached symmetrical, 25 (14 cranial duplication, 2 dipygus, 2 dicephalus dipygus, 5 thoracopagus, and 2 pygopagus); attached asymmetrical, 6 (1 parasitic dipygus, 4 notomelia, and 1 pygomelia); and miscellaneous, 4. The four acardii were holoacardii amorphi covered with skin and hair. Cranial duplication was subdivided into five types based on the number of eyes and ears. In symmetrical twins the anterior part of the body was affected in 16 out of 25 cases (64%), the posterior part in 2 out of 25 (8%) and both the anterior and posterior in 7 out of 25 (28%). All were mirror-image duplicates on the various planes, and in some the internal organs had a center of symmetry. All four notomeli calves were female. Among the miscellaneous duplications, three were of the genital organs (male 1, and female 2) and one was a spinal cord duplication.