Reduced satellite cell density and myogenesis in Wagyu compared with Angus cattle as a possible explanation of its high marbling.

Mechanisms responsible for excellent marbling in Japanese black cattle, Wagyu, remain to be established. Because both muscle cells and intramuscular adipocytes are developed from mesenchymal progenitor cells during early muscle development, we hypothesized that intramuscular progenitor cells in Wagyu cattle have attenuated myogenic capacity in favor of adipogenesis, leading to high marbling but reduced muscle growth. Biceps femoris muscle biopsy samples were obtained from both Angus (n=3) and Wagyu (n=3) cattle at 12 months of age. Compared with Angus, the density of satellite cells was much lower in Wagyu muscle (by 45.8±10%, P<0.05). Consistently, the formation of myotubes from muscle-derived progenitor cells was also lower (by 64.2±12.9%, P<0.05), but adipogenic capacity was greater in Wagyu. The average muscle fiber diameter was larger in Wagyu (by 23.9±6.8%, P=0.089) despite less muscle mass, suggesting less muscle fiber formation in Wagyu compared with Angus cattle. Because satellite cells are derived from fetal myogenic cells, the reduction in satellite cell density together with lower muscle fiber formation suggests that myogenesis was attenuated during early muscle development in Wagyu cattle. Given the shared pool of mesenchymal progenitor cells, the attenuated myogenesis likely shifts progenitor cells to adipogenesis during early development, which may contribute to high intramuscular adipocyte formation in Wagyu cattle.

Enhanced mitogenesis in stromal vascular cells derived from subcutaneous adipose tissue of Wagyu compared with those of Angus cattle.

Japanese Wagyu cattle are well known for their extremely high marbling and lower subcutaneous adipose tissue compared with Angus cattle. However, mechanisms for differences in adipose deposition are unknown. The objective of this paper was to evaluate breed differences in the structure of subcutaneous adipose tissue, adipogenesis, and mitogenesis of stromal vascular (SV) cells between Wagyu and Angus cattle. Subcutaneous biopsy samples were obtained from 5 Wagyu (BW = 302 ± 9 kg) and 5 Angus (BW = 398 ± 12 kg) heifers at 12 mo of age, and samples were divided into 3 pieces for histological examination, biochemical analysis, and harvest of SV cells. Adipogenesis of SV cells was assessed by the expression of adipogenic markers and Oil Red-O staining, while mitogenesis was evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium dromide) test, phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB; AKT). Based on histological analysis, Wagyu had larger adipocytes compared with Angus. At the tissue level, protein expression of peroxisome proliferator-activated receptor γ (PPARG) in Wagyu was much lower compared with that of Angus. Similarly, a lower mRNA expression of PPARG was found in Wagyu SV cells. No significant difference was observed for the zinc finger protein 423 (ZNF423) expression between Wagyu and Angus. As assessed by Oil Red-O staining, Wagyu SV cells possessed a notable trend of lower adipogenic capability. Interestingly, higher mitogenic ability was discovered in Wagyu SV cells, which was associated with an elevated phosphorylation of ERK1/2. There was no difference in AKT phosphorylation of SV cells between Wagyu and Angus. Moreover, exogenous fibroblast growth factor 2 (FGF2) enhanced mitogenesis and ERK1/2 phosphorylation of SV cells to a greater degree in Angus compared with that in Wagyu. Expression of transforming growth factor ß 3 (TGFB3) and bone morphogenetic protein 2 (BMP2) in Wagyu SV cells was lower than that of Angus, providing potential clues for breed differences on proliferation of SV cells in these two cattle breeds. The results of this study suggest that subcutaneous adipose-derived SV cells of Wagyu possess a lower trend of adipogenesis but higher mitogenesis compared with those of Angus.

Biosecurity and biocontainment in alpaca operations.

Biosecurity on South American camelid operations involves both external and internal measures to prevent the introduction and spread of disease. External biosecurity involves practices and techniques directed at the prevention of entry of new diseases into a group of animals. Internal biosecurity or biocontainment, involves practices and techniques that are directed at the prevention or spread of disease within an existing group of animals. External biosecurity is particularly important in North America camelid operations due to the extensive movement of animals for breeding or show purposes. Internal biosecurity typically involves this the prevention and treatment of failure of passive transfer, maintenance of proper nutrition and housing, and the implementation of an appropriate vaccination program for endemic or relevant diseases. Attention to appropriate cleaning and disinfection procedures related to housing, feeding, and treatment equipment is important for the maintenance of both internal and external biosecurity practices. This paper discusses various risk factors associated with the control of infectious disease in the context of external and internal biosecurity measures in camelids operations.

Age-related innate immune response in calves to Babesia bovis involves IL-12 induction and IL-10 modulation.

There is a strong innate immunity in calves to infection with Babesia bovis. Interleukin (IL)-12 and IL-10 have been shown in vitro to be important immunoregulatory cytokines. Here we demonstrate in vivo that the protective innate response in young calves to infection with virulent B. bovis involves the early appearance of IL-12 and interferon-gamma (IFN-gamma) transcripts in the spleen. In contrast, IL-12 and IFN-gamma mRNA expression in the spleens of adult cattle that succumbed to the infection was delayed and depressed and occurred within the context of IL-10 expression. Also in contrast with calves, there was no detectable antibody response before death in adults. A vigorous CD8+ T-cell expansion occurred in the spleens of both calves and adults.

IL-4 and IL-10 inhibition of IFN-gamma- and TNF-alpha-dependent nitric oxide production from bovine mononuclear phagocytes exposed to Babesia bovis merozoites.

The requirement for IFN-gamma and/or TNF-alpha as co-stimulants with Babesia bovis merozoites for nitric oxide (NO) production was examined, as well as the regulatory role of IL-4 and IL-10. Purified B. bovis merozoites did not induce the production of NO in undifferentiated monocytes without addition of exogenous IFN-gamma and TNF-alpha unless the monocytes taken ex vivo were producing TNF-alpha endogenously. Under the latter condition, the NO production resulting from merozoite stimulation remained IFN-gamma-dependent. There was no evidence for endogenous synthesis of TNF-alpha in monocyte-derived macrophages (MDM), and merozoites alone were incapable of inducing TNF-alpha mRNA in MDM. However, while merozoites plus IFN-gamma induced TNF-alpha mRNA expression in MDM, NO was not produced. Both IL-4 and IL-10 inhibited expression of iNOS and production of NO in merozoite-stimulated monocytes.

Bovine neonatal immunology.

The majority of early, in utero immune development occurs independent of antigen exposure. Only later during development can a fetus respond to antigens, and even then the response depends on the stage of fetal development and the nature of the antigen. At birth, the neonate is rapidly exposed to large numbers of potential pathogens. Although immunocompetent, the neonate is immunonaive and dependent on passively acquired maternal immunoglobulins, immune cells, and other substances from colostrum for protection. Neonates that suffer failure of passive transfer of maternal immunoglobulins may be at increased risk for disease; however, many other factors interact in conjunction with the level of passively acquired immunoglobulin to determine the occurrence of disease. These include, but are not limited to, management, environment, hygiene, infection pressure, virulence of organisms, and antibody specificity. In addition to immunoglobulins, colostrum contains large numbers of immune cells and cytokines. It is thought that the primary role for the cellular component of colostrum is to interact with the development of local immunity and to modulate active immunization of the neonatal intestine. In particular, T lymphocytes are thought to transfer immune functions and secrete cytokines. Although most of the major cytokines have been identified in colostrum and milk, their biologic effects on the neonate have yet to be determined.

The age-related immunity in cattle to Babesia bovis infection involves the rapid induction of interleukin-12, interferon-gamma and inducible nitric oxide synthase mRNA expression in the spleen.

Young calves possess a strong innate immunity against Babesia bovis infection that lasts for approximately 6 months after birth and is abrogated with the removal of the spleen. This immunity is characterized as cellular involving a soluble mediator. Nitric oxide has been implicated by virtue of its babesiacidal affects in vitro, but questioned to be as effective in vivo, due to its ability to downregulate type-1 immunity. Spleen cells were obtained from 4-month-old calves and adult steers and processed for monitoring cytokine and inducible nitric oxide synthase (iNOS) mRNA expression during the response to initial B. bovis infection. The data provided evidence of a transient role for nitric oxide in innate immunity, characterized by brief iNOS induction in the spleen of calves that was not detectable in the spleens of adults. The iNOS message followed the early induction of interleukin (IL)-12 and interferon (IFN)-gamma message in calves. The induction of IL-12 and IFN-gamma message in adults was delayed until IL-10 message was induced. Transformation growth factor-beta mRNA expression levels were greater in spleen cells from adults early in infection and then declined, whereas expression levels increased in spleen cells from calves later in the infection process. Together, the data support the concept of 'first come, first serve' cytokine influence over cellular activities, the importance of a type-1 response in the control of an initial infection and the need for tight regulation in order to prevent pathology associated with over production of nitric oxide and inflammatory cytokines.

Effects of tallow on the energy metabolism of wethers fed barley finishing diets.

A balance trial was conducted to titrate the effects of tallow on the energy metabolism of wethers fed barley finishing diets. Six dietary levels of tallow (0, 2, 4, 6, 8, or 10%) in a barley finishing diet were fed to six crossbred wethers (35+/-1.1 kg) in a randomized complete block design. Diets were 73% barley, 10% tallow and(or) bentonite, 10% alfalfa pellets, and 7% supplement. There was no effect of tallow level on OM intake (1,103.1+/-51 g/d), OM digestibility (84+/-0.9%), GE digestibility (83+/-1.1%), or cell solubles digestibility (84.2+/-1.2%). The level of tallow quadratically decreased ADF digestibility (P < 0.05), methane emissions, and methane energy as a percentage of GE P < 0.01). There were linear increases in dietary GE (megacalories per kilogram of OM [P < 0.01]), dietary DE (megacalories per kilogram of OM [P < 0.05]), and dietary ME (megacalories per kilogram of OM [P < 0.01]), as dietary tallow increased. Numbers of ruminal protozoa (Entodinium spp. and Polyplastron sp.) decreased linearly (P < 0.05) with increased level of tallow. The energy value of tallow (calculated by difference) was low. The total-tract fatty acid digestibility of tallow was calculated by linear regression, without intercept, after accounting for the fatty acids digested from the base diet (0% tallow fed to a wether in a period). Fatty acids of the same carbon length were pooled for the regression analysis. All linear regressions were significant (P < 0.10) indicating no effect of tallow level on fatty acid digestibility. Lauric acid had low digestibility. The high digestibility of all C16 (89%) and C18 (104%) fatty acids suggests an effect of tallow on endogenous and microbial fatty acid excretion. Fatty acid digestibility was probably a minor contributor to the low energy content of tallow, calculated by difference, in these diets.

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue.

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97. 6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.

Thyroid hormone coordinates respiratory control maturation and adenine nucleotide translocator expression in heart in vivo.

BACKGROUND: The signal transduction mechanism linking mitochondrial ATP synthesis with cytosolic ATP utilization in heart changes during postnatal development in vivo. This maturational process occurs in parallel with accumulation of mitochondrial adenine nucleotide translocator (ANT), which provides a possible site for respiratory control. We postulated that thyroid hormone regulates these maturational processes. METHODS AND RESULTS: We used (31)P MR spectroscopy to determine the relationship between myocardial high-energy phosphates, phosphocreatine, and ADP and oxygen consumption (MVO(2)) during epinephrine stimulation in 32- to 40-day-old lambs thyroidectomized after birth (THY) and age-matched controls. Steady-state protein and mRNA levels for ANT isoforms and beta-F(1)-ATPase were assessed from left ventricular tissues by Western and Northern blotting. With greater doses of epinephrine, THY attained lower peak MVO(2) than controls (P:<0.05). Controls maintained high-energy phosphate levels, unlike THY, which demonstrated significantly decreased phosphocreatine/ATP and increased cytosolic ADP despite lower peak MVO(2). No significant differences in beta-F(1)-ATPase protein or mRNA occurred between groups. However, ANT isoform mRNA levels were 2-fold greater and protein levels 4-fold greater in control hearts. CONCLUSIONS: These data imply that the maturational shift away from ADP-mediated respiratory control is regulated by thyroid hormone in vivo. Specific thyroid-modulated increases in ANT mRNA and protein imply that this regulation occurs in part at a pretranslational level.

Flow cytometric analysis of an immunodeficiency disorder affecting juvenile llamas.

The present study was undertaken to characterize the immune system of llamas and alpacas and establish the basis for an immunodeficiency disorder affecting juvenile llamas. Flow cytometric (FC) analysis of the immune system with a panel of monoclonal antibodies (mAbs) revealed the immune system of llamas and alpacas is similar in leukocyte subset composition to that in ruminants. Peripheral blood mononuclear cells in adults are comprised of surface immunoglobulin (sIg(+)) B-cells (31%+/-8 S.D.), alphabeta T-cells (27%+/-12 S.D.), WC1(+) gammadelta T-cells (16%+/-11 S.D.), and 5-16% monocytes. In contrast to cattle, goats, and sheep, however, the frequency of WC1(+) gammadelta T-cells is not high in juveniles but similar to the frequency in adults. Also, sIg(+) B-cells are present in high concentration in juveniles (43%+/-11 S.D. ). Expression of major histocompatibility class II molecules on resting T-cells was low or absent. Comparative analysis of peripheral blood lymphocyte composition in normal juvenile llamas and llamas presenting with the signs of the juvenile llama immunodeficiency syndrome (JLIDS) revealed the concentration of B-cells is extremely low (1-5%) in affected animals. The findings suggest JLIDS is attributable to an autosomal recessive genetic defect in the development of B-cells.

Prediction of serum IgG1 concentration in beef calves based on age and serum gamma-glutamyl-transferase activity.

This study examined the relationship between serum gamma-glutamyl-transferase (GGT) activity and passive transfer status in beef calves less than 18 days of age. Immunoglobulin G1 (IgG1) concentrations were measured in 69 commercial beef calves between the ages of 24 and 72 hours. GGT activities were then measured in these same calves at various ages between 3 and 18 days of age. Models were developed predicting serum IgG1 concentration as a function of calf age and serum GGT activity. Minimal association was present between initial serum IgG1 concentration and serum GGT activity when all calves less than 18 days of age were considered (r2 = 0.065). When the study population was restricted to calves less than 8 days of age, the fit of the developed model was greatly improved (r2 = 0.438). Serum GGT activity has no apparent advantage relative to other assay procedures for predicting passive transfer status in beef calves. If serum GGT activity is to be used to assess passive transfer status in beef calves, application of this procedure should be restricted to calves less than 8 days of age.

Detection of low serum immunoglobulin concentrations in clinically ill calves.

The sensitivity, specificity, and accuracy of classification of 4 tests for failure of passive transfer (FPT) were examined in clinically ill neonatal calves. Comparisons were made with serum IgG1 concentrations determined by radial immunodiffusion. Serum samples were obtained from 27 clinically ill calves < or = 21 days of age. The results of 4 commonly used assays, the sodium sulfite turbidity test, the zinc sulfate turbidity test, refractometry, and the serum gamma-glutamyl transferase (GGT) activity test, were compared with radial immunodiffusion determinations of serum IgG1 concentration. Serum GGT activity using a 50 IU/L threshold resulted in correct classification of the highest percentage of calves (93%) with regard to their passive transfer status. The sodium sulfite test with a 1+ end point and refractometry using a 5.5 g/dL end point resulted in correct classification of 85% of the calves studied. When using the sodium sulfite test, the 2+ and 3+ test end points had lower specificity, 0.58 and 0.00, respectively, than the 1+ end point. This loss in specificity resulted in misclassification of calves with adequate serum immunoglobulin concentrations as having FPT. The zinc sulfate turbidity test was inadequately specific (0.33) and resulted in misclassification of 33% of calves.

Conservative management of a ruptured gastrocnemius muscle in a male llama.

Rupture of the gastrocnemius muscle was diagnosed in an obese llama using physical examination and ultrasound imaging. Conservative therapy consisting of the use of a cast to immobilize the affected limb permitted the muscle to heal. Only mild, residual gait abnormalities were observed on follow-up examination.

Facial fibrosarcoma in two cows.

Fibrosarcoma is a common tumor affecting bone, but is uncommon in cows. Reported here are two bovine cases of facial fibrosarcoma that were very invasive.

Use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs.

OBJECTIVE: To develop an algorithm for predicting passive transfer status of lambs of various ages, using the lamb's age and serum gamma-glutamyltransferase (GGT) activity. DESIGN: Prospective study. ANIMALS: 51 Suffolk, Columbia, and crossbred lambs from 1 to 16 days old. PROCEDURE: Serum was obtained from all lambs. Serum GGT activity was measured, using a commercially available kit. Serum IgG concentration was determined by use of radial immunodiffusion. Day-1 serum IgG concentration was estimated from sample IgG concentration, lamb age, and the published 14-day half-life of IgG in lambs. Stepwise multivariate regression models were developed to estimate day-1 serum IgG concentration as a function of the natural logarithm of serum GGT activity (In[GGT]) and natural logarithm of lamb age (In[age]) at the time of sampling. These regression models were then used to calculate serum GGT activities that were equivalent to various day-1 IgG concentrations in lambs of various ages. RESULTS: In(GGT) and In(age) were significantly associated with estimated day-1 IgG concentration. Day-1 serum IgG concentration could be predicted using the formula: IgG = -7,686 + 1,366(In[GGT]) + 1,199(In[age]). The model was moderately accurate in predicting serum IgG concentration (R2 = 0.52). CLINICAL IMPLICATIONS: Serum GGT activity can be used to assess passive transfer status of lambs.