Comparison of mathematical and comparative slaughter methodologies for determination of heat production and energy retention in broilers.

Understanding factors affecting ME availability for productive processes is an important step in optimal feed formulation. This study compared a modelling methodology with the comparative slaughter technique (CST) to estimate energy partitioning to heat production and energy retention (RE) and to investigate differences in heat dissipation. At hatch, 50 broilers were randomly allocated in one of 4 pens equipped with a precision feeding station. From day 14 to day 45, they were either fed with a low-ME (3,111 kcal/kg ME) or a high-ME (3,383 kcal/kg ME) diet. At day 19, birds were assigned to pair-feeding in groups of 6 with lead birds eating ad libitum (100%) and follow birds eating at either 50, 60, 70, 80, or 90% of the paired lead's cumulative feed intake. Heat production and RE were estimated by CST and with a nonlinear mixed model explaining daily ME intake (MEI) as a function of metabolic BW and average daily gain (ADG). The energy partitioning model predicted MEI = (145.10 + u) BW0.83 + 1.09 × BW-0.18 × ADG1.19 + Îµ. The model underestimated heat production by 13.4% and overestimated RE by 22.8% compared with the CST. The model was not able to distinguish between net energy for gain values of the diets (1,448 ± 18.5 kcal/kg vs. 1,493 ± 18.0 kcal/kg for the low-ME and high-ME diet, respectively), whereas the CST found a 148 kcal/kg difference between the low-ME and high-ME diets (1,101 ± 22.5 kcal/kg vs. 1,249 ± 22.0 kcal/kg, respectively). The estimates of the net energy for gain values of the 2 diets decreased with increasing feed restriction. The heat increment of feeding did not differ between birds fed with the low- or high-ME diet (26% of MEI). Additional measurements on heat dissipation, physical activity, and immune status indicated that the energetic content of the diet and feed restriction affect some parameters (shank temperature, feeding station visits) but not others (leukocyte counts, heterophil to lymphocyte ratio, and immune cell function).

Mallards (Anas platyrhynchos) and wastewater ponds, Part II: Developmental, physiological, morphological and behavioural effects of ingestion of secondary clarified effluent water.

Rather than migrating, mallard ducks may choose to overwinter in northern cities on open-water thermal refuges, such as municipal wastewater treatment ponds, which in Edmonton, Canada, stay ≥10°C during frigid winter months. Refuging mallards spend appreciable time daily on these ponds and hydrate using secondary clarified municipal wastewater (SCEW). We aimed to determine if SCEW ingestion affected mallard health. To this end, we gavaged newly hatched mallards (domesticated Pekin strain) over their first month with SCEW, as well as water representing negative and positive controls (municipal tap water, and the primary active ingredient from birth control pills, 17α-ethinyl estradiol (EE2), respectively). The gavage of SCEW did not affect mass of the body, liver, spleen or heart, but was associated with small increases in beak and wing chord length. In the positive control, EE2 gavage caused similar responses, but also increased tarsus and phallus length. The increases likely owed to the stimulatory effects of estrogenic substances on bone and phallus development. For the biotransformation enzyme CYP2H1, gene expression was numerically increased by both SCEW and EE2. In terms of behavior, SCEW and EE2 gavage reduced two infrequently detected behaviours, pecking and resting alone. Our results suggest that SCEW ingestion would be unlikely to cause any overt health effects in adults, but may evoke subtle, covert effects nevertheless.

Contributions of transferrin to acute inflammation in the goldfish, C. auratus.

Transferrin is an evolutionary conserved protein that in addition to having a critical role in iron transport also has been shown to have a crucial role in host defence, by depriving iron from invading pathogens. Recently cleaved transferrin products was shown to activate macrophages in vitro. We now use an in vivo model of self-resolving peritonitis in goldfish, coupled with gene expression and protein analysis to evaluate the contributions of cleaved transferrin to acute inflammation. We show, for the first time, that cleaved transferrin products are produced in vivo early during an acute inflammatory response. These cleaved transferrin fragments were produced during pathogen-induced, but not sterile, inflammation. Both macrophages and neutrophils were able to contribute to transferrin cleavage. However, only macrophages contributed to this innate process through inducible expression of transferrin. The appearance of transferrin cleavage products in vivo correlated with the influx of leukocytes but did not necessarily correlate the induction of robust respiratory burst and nitric oxide responses. Overall, this study adds to a growing body of work highlighting the role of transferrin as an immune regulator during acute inflammation. Given the significant conservation of this and related molecules, these findings have potentially broad implications for host defences and inflammation control across evolution.

The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation.

The objective of the current study was to determine the effects of adding 3-nitrooxypropanol to the diet of lactating Holstein cows on methane emissions, rumen fermentation, ruminal microbial profile, and milk production. Twelve ruminally cannulated Holstein cows in midlactation were used in a crossover design study with 28-d periods. Cows were fed a diet containing 38% forage on a dry matter basis with either 2,500 mg/d of 3-nitrooxypropanol (fed as 25 g of 10% 3-nitrooxypropanol on silicon dioxide) or 25 g/d of silicon dioxide (control). After a 21-d diet adaptation period, dry matter intake (DMI) and milk yield were recorded daily. Rumen fluid and digesta were collected on d 22 and 28 for volatile fatty acid analysis and microbial profiling. Enteric methane emissions were measured on d 23 to 27 using the sulfur hexafluoride tracer gas technique. Feeding 3-nitrooxypropanol did not affect DMI; however, methane production was reduced from 17.8 to 7.18 g/kg of DMI. No change in milk or milk component yields was observed, but cows fed 3-nitrooxypropanol gained more body weight than control cows (1.06 vs. 0.39 kg/d). Concentrations of total volatile fatty acids in ruminal fluid were not affected by treatment, but a reduction in acetate proportion and a tendency for an increase in propionate proportion was noted. As such, a reduction in the acetate-to-propionate ratio was observed (2.02 vs. 2.36). Protozoa counts were not affected by treatment; however, a reduction in methanogen copy count number was observed when 3-nitrooxypropanol was fed (0.95 vs. 2.69 × 10(8)/g of rumen digesta). The data showed that feeding 3-nitrooxypropanol to lactating dairy cows at 2,500 mg/d can reduce methane emissions without compromising DMI or milk production.

Triennial Growth Symposium--Novel roles for vitamin D in animal immunity and health.

Recent years have seen significant advances in the generation, validation, and implementation of nutritional supplements for food production animals. Examination of their impact on animal performance and health requires collaboration among animal scientists, nutritionists, biochemists, immunologists, veterinarians, and others. Each provides a unique perspective on the mechanisms of action, short and long-term impacts, and most effective strategies for implementation into continuously evolving industrial practices. In this review we provide a comparative immunology perspective on the impact of vitamin D on animal performance and health, describe the differential contributions of vitamin D3 and of a commercial hydroxylated version of vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3 or HyD) to swine immunity, and highlight recent advances in the technologies that can be used to dissect the cellular and molecular mechanisms that impact production animal immunity and health. Among others, we pay particular attention to how these novel approaches help decrease the variability often observed in immune-associated datasets. From a practical perspective, this is critical for evaluation of in vivo effects for this nutritional supplement as small but meaningful changes to specific immune responses are typical under normal physiological conditions. Furthermore, as the range of reagents and technologies expands for comparative animal models, it is imperative that continued efforts are placed on the capacity to compare results across different experimental platforms.

Moderate decreases in the forage-to-concentrate ratio before feed restriction and increases thereafter independently improve the recovery from a feed restriction insult in beef cattle.

The objective of this study was to determine if the forage-to-concentrate ratio (F:C) of diets fed prior to and during (PRE) feed restriction (FR) and diets fed post-FR (POST) affect the recovery for DMI, ruminal fermentation, and short-chain fatty acid (SCFA) absorption following FR. Twenty ovariectomized and ruminally cannulated Angus × Hereford heifers were used in this study and were fed (ad libitum) either a high forage (HF; F:C = 92:8) or a moderate forage (MF; F:C = 60:40) diet for 19 d. Heifers were then exposed to a 5-d FR period where feed was restricted to 25% of ad libitum intake relative to that measured during the previous 5 d. After FR, heifers were provided feed ad libitum with one half of the HF and MF heifers receiving the HF or MF diet during the 3-wk recovery period (REC1, REC2, and REC3). This resulted in 4 treatment combinations separated over time (PRE-POST): HF-HF, HF-MF, MF-HF, and MF-MF. The PRE × POST interaction was not significant for any of the measured variables, and the PRE × POST × period interaction was only significant for the molar proportion of ruminal butyrate. For heifers fed HF PRE, DMI increased from REC1 to REC3 whereas DMI did not differ among periods for heifers fed MF PRE (PRE × period, P = 0.045). The duration that pH < 5.5 (PRE × POST; P = 0.003) was numerically greater during REC1 for heifers fed HF than MF PRE (191 vs. 98 min/d), with duration decreasing from REC1 to REC2 for heifers fed HF PRE. Total ruminal SCFA concentration and absorption rate were not affected by the diet fed PRE (P > 0.05) or period (P > 0.05). For heifers fed MF POST, DMI increased from REC1 to REC3 whereas DMI did not differ among POST periods for heifers fed HF POST (POST × period, P = 0.033). The duration that ruminal pH was <5.5 was greater for heifers fed MF than HF POST (274.9 vs. 14.1 min/d; POST × period, P < 0.001) with MF heifers decreasing duration from REC1 to REC2 whereas duration did not differ among periods for HF. Ruminal SCFA concentration and rate of absorption were not affected (P > 0.05) by diet fed POST. It is concluded that feeding a MF diet prior to and during FR improves the recovery response for DMI. Irrespective of the prefeeding, however, a HF diet is beneficial in the POST-restriction period because it most effectively alleviated ruminal pH reduction and hastened DMI recovery.

Feed restriction reduces short-chain fatty acid absorption across the reticulorumen of beef cattle independent of diet.

The objective of this study was to evaluate the effects of the forage-to-concentrate ratio (F:C) of diets fed before and during short-term feed restriction (FR) on rumen fermentation, absorptive capacity of the reticulorumen, and apparent total tract digestibility. Twenty ovariectomized and ruminally cannulated Angus × Hereford heifers were blocked by BW and individually penned in box stalls (9 m(2)), having free access to water throughout the study. Heifers were randomly assigned to 1 of 2 dietary treatments, receiving either a high forage diet (HF; F:C of 92:8) or a moderate forage diet (MF; F:C of 60:40). Diets were fed ad libitum for 14 d before 5 d of baseline measurements (BASE) followed by 5 d of FR where heifers were restricted to 25% of ad libitum DMI relative to BASE. Dry matter intake was measured daily and ruminal pH was recorded every 2 min throughout the study. Ruminal fluid and blood samples were collected on d 3 of BASE and FR whereas short-chain fatty acid (SCFA) absorption was assessed in vivo using the isolated washed reticulorumen technique on d 5 of BASE and FR. Indigestible NDF was used as a marker to estimate apparent total tract digestibility. Diet × period interactions (P = 0.030 and 0.025) were detected for DMI and ruminal SCFA concentration, respectively. The interaction was the result of greater DMI and numerically greater SCFA concentration for MF than HF during BASE, with a reduction observed for both during FR, although treatment effects were no longer present. Period effects (BASE vs. FR) but not treatment effects (P > 0.05) were detected for mean ruminal pH (P < 0.001) and the total SCFA absorption rate (mmol/h; P = 0.038). During BASE, mean pH was reduced (6.4 vs. 6.9) and the SCFA absorption rate was greater relative to FR (674.5 vs. 554.8 mmol/h). Diet (P < 0.001) and period (P < 0.001) effects were detected for DM and OM digestibility with greater digestibility occurring for heifers fed MF than HF (70.5 vs. 63.3% for DM and 73.0 vs. 66% for OM) and greater digestibility during FR than BASE (69.5 vs. 64.3% for DM and 71.7 vs. 67.2% for OM). During FR, NDF digestibility was also greater than during BASE (P < 0.001; 62.4 vs. 55.8%). The effect of FR on serum NEFA differed by diet (diet × period, P < 0.001) with NEFA being greater for heifers fed HF than MF during FR (474.4 vs. 377.7 µEq/mL, respectively) with no differences observed between HF and MF during BASE. It can be concluded that severe short-term FR had a negative impact on ruminal SCFA absorption and energy balance and that altering the F:C of the diet does not mitigate these effects.

Recovery of absorptive function of the reticulo-rumen and total tract barrier function in beef cattle after short-term feed restriction.

Our objective was to determine if the severity of short-term feed restriction (FR) affects the timeline for recovery of the absorptive function of the reticulo-rumen and barrier function of the total gastrointestinal tract in beef cattle. Eighteen ruminally cannulated and ovariectomized Angus × Hereford heifers were housed in individual pens. Heifers were blocked by initial BW into 3 blocks and, within block, randomly assigned to 1 of 3 treatments that differed in the severity of FR: heifers were restricted to 75, 50, or 25% of ad libitum intake. Treatments were imposed during a 5-d period of FR followed by 3 consecutive wk of recovery (REC1, REC2, and REC3). Throughout the experiment heifers were fed the same diet (60% forage:40% concentrate) for ad libitum intake (except during FR) and water was available at all times. Dry matter intake was measured daily and ruminal pH was recorded every 2 min during FR and recovery periods. Ruminal fluid and blood samples were collected on d 3 of the FR and d 5 of REC1 and REC3. Short-chain fatty acid (SCFA) absorption rates were evaluated on the last day of FR, REC1, and REC3 using the temporarily isolated and washed reticulo-rumen technique. On d 2 of FR and d 4 of REC1 and REC3, a 1 L solution of Cr-EDTA (180 mM) was dosed into the rumen followed by 48 h of total urine collection. Dry matter intake (% BW) increased rapidly in REC1 for heifers restricted to 75 and 50%; however, heifers restricted to 25% needed at least 2 wk to recover (treatment × period; P < 0.001). Regardless of the severity of FR, the duration that pH < 5.5 was the highest during REC1 (period P < 0.001). However, an interaction was found for the acidosis index, with pH × min/kg of DMI being greatest in heifers restricted to 25% on d 1 of the recovery period. A treatment × period interaction was found for the absolute absorption rate (mmol/h) of total SCFA (P = 0.009). The total SCFA absorption rate was not different for heifers restricted to 75 and 50% across periods, whereas an increase from FR and REC1 to REC3 was detected for heifers restricted to 25% of ad libitum intake. A treatment effect was observed for urinary Cr output (P = 0.027) indicating that heifers previously restricted to 25% of ad libitum intake had greater Cr excretion in urine during FR and recovery. This study indicates that severe FR negatively affects the time required for recovery of reticulo-rumen absorptive function and total tract barrier function. Another important finding is that regardless of severity, FR increases risk for ruminal acidosis when heifers have free access to feed after FR.

Short-term feed restriction impairs the absorptive function of the reticulo-rumen and total tract barrier function in beef cattle.

The objective of this study was to evaluate whether different severities of short-term feed restriction (FR) affect the absorptive function of the reticulo-rumen and total tract barrier function in beef cattle. Eighteen ruminally cannulated and ovariectomized Angus × Hereford heifers were blocked by BW into 3 blocks, with blocks conducted sequentially. Treatments were imposed during the 5-d FR period by restricting heifers to 75 (FR75), 50 (FR50) or 25% (FR25) of the ad libitum feed intake measured during a 5-d baseline period (BASE) occurring immediately before FR. Throughout the study, heifers were housed in individual pens (9 m(2)) and were fed the same diet (60% forage:40% concentrate) with free access to water. Dry matter intake was measured daily and ruminal pH was measured every 2 min throughout the study. Ruminal fluid and blood samples were collected on d 3 of the BASE and FR periods, and the temporarily isolated and washed reticulo-rumen technique was used to evaluate short-chain fatty acid (SCFA) absorption on d 5 of the BASE and FR periods. Total tract barrier function was evaluated starting on d 2 of the BASE and FR periods using a pulse dose of Cr-EDTA followed by 48 h of total urine collection. Data were analyzed using the Proc Mixed procedure of SAS with the fixed effects of block, treatment, period, and the treatment × period interaction, the random effect of cow nested in block with period included as a repeated measure. Dry matter intake did not differ among treatments during BASE but, as imposed by the experimental model, DMI during FR relative to BASE equated to 70, 49, and 25%, which was close to the targeted values of 75, 50, and 25% (treatment × period, P < 0.001). A treatment × period interaction (P < 0.001) was also detected for ruminal SCFA concentration with the concentration decreasing as the severity of FR increased, whereas there were no differences during BASE. Mean ruminal pH increased during FR with increasing severity of FR, but was not different during BASE (treatment × period, P < 0.001). Absorption of SCFA across the reticulo-rumen tended to decrease with increasing severity of FR (P = 0.08). For individual SCFA, acetate absorption (mmol/h) tended (P = 0.057) to be less for FR25 and FR50 when compared with FR75 and decreased (P = 0.05) by almost 70 mmol/h at FR25 and FR50 relative to BASE (322mmol/h). Heifers restricted to 25% (FR25) feed had greater urinary Cr recovery during FR than BASE, whereas no changes were detected for FR75 and FR50. This study indicates that moderate severities of short-term FR decrease the absorptive function of the reticulo-rumen, but more severe FR is required to compromise total tract barrier function in beef cattle.

Broiler egg storage induces cell death and influences embryo quality.

It is well known that egg storage reduces embryo performance, but the fundamental reasons for reduced embryo quality remain unclear. The objective of this study was to investigate possible cellular and molecular mechanisms that might reduce embryo quality after egg storage. Broiler hatching eggs were obtained from the Ross 308 broiler strain, divided into 2 groups, and stored (4 and 14 d) under the same temperature and humidity conditions. Samples of the eggs were used to assess embryo quality by determining daily embryo weight (wet and dry) from 4 to 21 d of incubation. To understand possible cellular and molecular mechanisms that might affect embryo quality, blastoderms (unincubated embryos) were isolated from a sample of eggs from each storage group, dissociated into single cells, and subjected to flow cytometry analysis to differentiate between viable, apoptotic, and necrotic cell populations. Quantitative real-time PCR analysis was used to compare the expression of selected apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, Bcl-2-related ovarian killer gene, B-cell lymphoma 2 gene, and B-cell lymphoma xL gene) in blastoderms and embryos (6 d old after incubation). Data were analyzed by the MIXED model procedure of SAS (SAS Institute, Cary, NC), with significance set at P ≤ 0.05. After covariance analysis of initial egg weights, the results showed decreased daily embryo weights (wet and dry), an indication of decreased embryo quality that could affect hatch quality. In addition, a decrease in blastodermal cell viability was associated with an increased percentage of apoptotic cell deaths (P < 0.0001). Expression of pro-apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, and Bcl-2-related ovarian killer gene) were upregulated at the blastodermal level as the storage duration increased, but all genes were downregulated after 6 d of incubation. This suggests that an increase in egg storage duration could activate mechanisms of apoptotic cell death at the blastodermal level, which may be one of the molecular mechanisms that leads to reduced daily embryonic weight during incubation. Experimental controls capable of reducing the cellular and molecular mechanisms of egg storage should be used to increase embryo quality.

Influence of parent flock age on embryonic metabolism in modern turkey strains.

The metabolic response of some galliform embryos to embryonic heat production (EHP) and how incubation conditions have been adjusted to prevent overheating of embryos is well established in broiler breeders. However, the daily metabolic status of turkey embryos has not been studied or established in turkey embryos. The objectives of the current research were therefore to determine the respiratory (eggshell conductance, G) and metabolic status (EHP) of 2 modern turkey genetic strains [Hybrid (H) and Nicholas (N)] and 4 parent flock ages [young (Y, 30 wk), peak (P, 34 wk), mature (M, 55 wk), and old (O, 60 wk)] during incubation. To measure G, moisture loss from 15 eggs/genetic strain per flock age and saturated vapor pressure measured between the eggshell and its immediate environment were used. Daily embryonic O(2) consumption and CO(2) production rates were assessed 6 times each day from embryos of eggs (n = 11 eggs/genetic strain per flock age) incubated in individual metabolic chambers and were used to determined daily EHP. Data were analyzed using the mixed model procedure of SAS at P ≤ 0.05. The results showed that the G values (g/d per mmHg) were significantly different for the interaction between genetic strain and parent flock age (H × Y = 17.71, H × P = 17.53, H × M = 19.73, H × O = 26.46, N × Y = 16.70, N × P = 20.96; N × M = 25.47, N × O = 26.05; P = 0.0227). Daily EHP (mW) was higher in embryos from the O flock than in embryos from the Y flock during all days presented except at 8, 25, and 28 d of incubation (4 d: Y = 1.00, P = 0.93, M = 1.60, O = 1.75; 12 d: Y = 19.0, P = 20.0, M = 21.6, O = 23.4; 16 d: Y = 51.7, P = 60.5, M = 65.9, O = 70.8; 20 d: Y = 129, P = 146, M = 144, O = 155; 24 d: Y = 154, P = 188, M = 167, O = 180; 26 d: Y = 169, P = 199, M = 197, O = 230; and 27 d: Y = 231, P = 265, M = 288, O = 307; P < 0.05). The data showed that metabolic differences existed between embryos from flocks of different ages and that embryos from older flocks were metabolizing at a higher rate and could be subject to overheating, which requires further investigation. On the basis of the data, turkey eggs from flocks of different ages should be incubated separately for optimal physiological performance.

Dissociation of chicken blastoderm for examination of apoptosis and necrosis by flow cytometry.

We describe a method of isolating blastodermal cells for characterization through flow cytometry. Particular attention was placed on cell viability and integrity issues faced by conventional protocols. The method allowed us to examine mechanisms behind cellular death. Our protocol was optimized by the spatial resolution of the ImageStream multispectral imaging flow cytometer. Overall, the technique provides both quantitative and qualitative information on blastodermal cells. The methodology was applied to the current biological problem in which prolonged (14 d) versus short-term (4 d) layer egg storage reduces embryo viability. Data were obtained between 3 egg storage treatments (unstored, 4 d, and 14 d); the data were analyzed by the PROC MIXED model procedure of SAS at P < or = 0.05 and least squares means separated by the PDIFF procedure of SAS. The results showed that egg storage increases the rate of cell death by both apoptosis and necrosis. Importantly, our study showed higher percentages of necrosis and late apoptosis in long-term versus short-term stored eggs. The percentage of live cells decreased significantly when eggs were stored for 14 d (71.42 + or - 3.36%) compared with eggs stored for 4 d (83.58 + or - 2.15%). The percentage of early apoptotic cells was not significantly different between the 2 treatments. The percentage of necrotic cells and late apoptotic-necrotic cells was higher in eggs stored for 14 d (16.75 + or - 1.73%; 7.36 + or - 1.53%) versus eggs stored for 4 d (3.56 + or - 1.64%; 2.31 + or - 1.52%), respectively. This could negatively affect embryo survival because of the potential effect that necrosis has on surrounding tissue integrity. The technique will be particularly relevant in studies requiring single cells from chicken blastoderms or as a basis to characterize genes that regulate apoptosis in avian species.

Transcriptional regulation of hemopoiesis.

The regulation of blood cell formation, or hemopoiesis, is central to the replenishment of mature effector cells of innate and acquired immune responses. These cells fulfil specific roles in the host defense against invading pathogens, and in the maintenance of homeostasis. The development of hemopoietic cells is under stringent control from extracellular and intracellular stimuli that result in the activation of specific downstream signaling cascades. Ultimately, all signal transduction pathways converge at the level of gene expression where positive and negative modulators of transcription interact to delineate the pattern of gene expression and the overall cellular hemopoietic response. Transcription factors, therefore, represent a nodal point of hemopoietic control through the integration of the various signaling pathways and subsequent modulation of the transcriptional machinery. Transcription factors can act both positively and negatively to regulate the expression of a wide range of hemopoiesis-relevant genes including growth factors and their receptors, other transcription factors, as well as various molecules important for the function of developing cells. The expression of these genes is dependent on the complex interactions between transcription factors, co-regulatory molecules, and specific binding sequences on the DNA. Recent advances in various vertebrate and invertebrate systems emphasize the importance of transcription factors for hemopoiesis control and the evolutionary conservation of several of such mechanisms. In this review we outline some of the key issues frequently identified in studies of the transcriptional regulation of hemopoietic gene expression. In teleosts, we expect that the characterization of several of these transcription factors and their regulatory mechanisms will complement recent advances in a number of fish systems where identification of cytokine and other hemopoiesis-relevant factors are currently under investigation.

Characterisation of growth enhancing factor production in different phases of in vitro fish macrophage development.

We previously described the release of macrophage growth factor(s) (MGF) into culture supernatants (CCM) by a goldfish macrophage cell line (GMCL) and in vitro derived kidney macrophages (IVDKM). In this study, we report that IVDKM growth can be subdivided into three developmental phases, defined using both morphological and flow cytometric characteristics: a lag phase, a proliferative phase, and a senescence phase. Analysis of the growth inducing capabilities of CCM indicated that maximum activity was consistently found in supernatants isolated from IVDKM cultures during the proliferative phase of development. In contrast, CCM from the senescence phase proved to be poor inducers of macrophage growth. Overall, we identify a link between the seeding-CCM composition, the extent of IVDKM growth and the rate of entrance into a senescent state characterised by IVDKM apoptotic cell death. Use of IVDKM CCM obtained at the peak of macrophage growth maximised macrophage growth factor (MGF) activity, and prevented the introduction of negative regulators of IVDKM proliferation, which will contribute significantly to our MGF purification efforts. Furthermore, the collection of IVDKM, prior to their commitment into apoptotic pathways, will prove to be essential in the selection of specific cell subsets for studies of antimicrobial mechanisms of macrophages.

Generation and functional analysis of distinct macrophage sub-populations from goldfish (Carassius auratus L.) kidney leukocyte cultures.

Three distinct sub-populations of macrophages derived from goldfish kidney leukocyte cultures were generated and characterised. The sub-populations designated as R1, R2 and R3-type macrophages had distinct morphological, cytochemical and flow cytometric profiles, and also differed in their anti-microbial functions after activation with macrophage activation factors (MAF) and bacterial lipopolysaccharide (LPS). The R1-type macrophages were small cells that contained acid phosphatase, but lacked myeloperoxidase and non-specific esterase. The R2-type macrophages were morphologically similar to mature tissue macrophages of mammals, and were positive for acid phosphatase, myeloperoxidase and non-specific esterase. The R3-type macrophages were round cells with eccentrically placed nuclei and resembled mammalian monocytes. This sub-population stained for acid phosphatase, myeloperoxidase and non-specific esterase. The R2 and R3-type macrophages exhibited distinct functional responses after activation with MAF and/or LPS. R2-type macrophages were potent producers of nitric oxide, while R3-type macrophages produced little or no nitric oxide after activation with MAF and LPS. The R2 and R3-type macrophages also exhibited unique respiratory burst responses (ROI) after treatment with MAF and/or LPS. After treatment with MAF and LPS, activated R2 macrophages were primed for ROI after only 6 h of stimulation with the activating agents, and continued to exhibit a strong ROI response for an extended cultivation period (48 h). In contrast, activated R3-type macrophages showed an early ROI response (6 h after treatment with MAF and LPS), which decreased significantly by 48 h after treatment with the activating agents. Our results suggest that the analysis of the mechanisms of induction of fish anti-microbial responses may be dependent upon the concerted actions of functionally distinct macrophage sub-populations.

Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages.

We recently demonstrated that a goldfish macrophage cell line (GMCL) and primary in vitro-derived kidney macrophage (IVDKM) cultures contain three distinct macrophage subpopulations. Morphological, cytochemical, functional, and flow cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at distinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of mature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fluorescent dye and analysis of the data using the MODFIT software. Our results showed that IVDKM cultures proliferated non-synchronously, suggesting the presence of a temporal control mechanism regulating the number of cells entering the paths towards maturation. Such control is most evident during early progenitor proliferation and differentiation events. Our results showed that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observation of the mature macrophage-like subpopulation indicated that: 1) they appear to develop from both, the differentiation of monocyte-like cells, and direct differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage-like cells appeared to be capable of self-proliferation. Our results suggest the presence of alternate pathways of fish macrophage development other than the classical hematopoietic pathway.