The amino acid sensor methionyl-tRNA synthetase is required for methionine-induced milk protein synthesis in a domestic pigeon model.

This study was conducted to investigate whether methionyl-tRNA synthetase (MetRS) is a mediator of methionine (Met)-induced crop milk protein synthesis via the janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signalling pathway in breeding pigeons. In Experiment 1, a total of 216 pairs of breeding pigeons were divided into three groups (control, Met-deficient, and Met-rescue groups). In Experiments 2 and 3, forty pairs of breeding pigeons from each experiment were allocated into four groups. The second experiment included a control group and three MetRS inhibitor (REP8839) groups. The third experiment included a Met-deficient group, Met-sufficient group, REP8839 + Met-deficient group and REP8839 + Met-sufficient group. Experiment 1 showed that Met supplementation increased crop development, crop milk protein synthesis, the protein expression of MetRS and JAK2/STAT5 signalling pathway, and improved squab growth. Experiment 2 showed that crop development, crop milk protein synthesis and the protein expression of MetRS and the JAK2/STAT5 signalling pathway were decreased, and squab growth was inhibited by the injection of 1·0 mg/kg body weight REP8839, which was the selected dose for the third experiment. Experiment 3 showed that Met supplementation increased crop development, crop milk protein synthesis and the expression of MetRS and JAK2/STAT5 signalling pathway and rescued squab growth after the injection of REP8839. Moreover, the co-immunoprecipitation results showed that there was an interaction between MetRS and JAK2. Taken together, these findings indicate that MetRS mediates Met-induced crop milk protein synthesis via the JAK2/STAT5 signalling pathway, resulting in improved squab growth in breeding pigeons.

Pigeon during the Breeding Cycle: Behaviors, Composition and Formation of Crop Milk, and Physiological Adaptation.

Pigeon is an important economic poultry species in many countries. As an altricial bird, its growth and development are largely reliant on pigeon milk produced by the crop tissue in the first week. During the breeding cycle, pigeons undergo a series of behavioral changes. Pigeon milk is generally characterized by having high concentrations of proteins and lipids, and a complicated regulatory network is involved in the milk formation. Hormones, especially prolactin, could promote the proliferation of crop epidermal cells and nutrient accumulation. The expression of target genes associated with these important biological processes in the crop epidermis is affected by non-coding RNAs. Meanwhile, signaling pathways, such as target of rapamycin (TOR), Janus kinase/signal transducer and activator of transcription proteins (JAK/STAT), protein kinase B (Akt), etc., influence the production of crop milk by either enhancing protein synthesis in crop cells or inducing apoptosis of crop epidermal cells. In order to adapt to the different breeding periods, pigeons are physiologically changed in their intestinal morphology and function and liver metabolism. This paper reviews the behaviors and physiological adaptations of pigeon during the breeding cycle, the composition of pigeon crop milk, and the mechanism of its formation, which is important for a better understanding of the physiology of altricial birds and the development of artificial crop milk.

Chemical composition of pigeon crop milk and factors affecting its production: a review.

Pigeons are important commercial poultry in addition to being ornamental birds. In 2021, more than 111 million pairs of breeding pigeons were kept in stock and 1.6 billion squabs were slaughtered for meat in China. However, in many countries, pigeons are not domestic birds; thus, it is necessary to elucidate the factors involved in their growth and feeding strategy due to their economic importance. Pigeons are altricial birds, so feedstuffs cannot be digested by squabs, which instead are fed a mediator named pigeon crop milk. During lactation, breeding pigeons (both female and male) ingest diets and generate crop milk to feed squabs. Thus, research on squab growth is more complex than that on chicken and other poultry. To date, research on the measurement of crop milk composition and estimation of the factors affecting its production has not ceased, and these results are worth reviewing to guide production. Moreover, some studies have focused on the formation mechanism of crop milk, reporting that the synthesis of crop milk is controlled by prolactin and insulin-activated pathways. Furthermore, the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) pathway, target of rapamycin (TOR) pathway and AMP-activated protein kinase (AMPK) pathway were also reported to be involved in crop milk synthesis. Therefore, this review focuses on the chemical composition of pigeon crop milk and factors affecting its production during lactation. This work explores novel mechanisms and provides a theoretical reference for improving production in the pigeon industry, including for racing, ornamental purposes, and production of meat products.

Analyses of circRNAs profiles of the lactating and nonlactating crops in pigeon (Columba livia).

Pigeon has the specific biological ability to produce pigeon milk (also known as crop milk) by its crop. Circular RNAs (circRNAs) are important noncoding RNAs acting as the sponges of miRNAs, but the molecular mechanism of circRNAs regulating crop milk production has not been reported in pigeon. We compared expression profiles of crops during lactating and nonlactating crops, and networks of competing endogenous RNAs (ceRNAs) were constructed. The results showed a total of 8,723 circRNAs were identified, and there were 770 differentially expressed circRNAs (DECs) between these two different periods of crops. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the host genes of DECs were enriched in GnRH, MAPK, Insulin, Wnt, and AMPK signaling pathways. Furthermore, gga_circ_0000300 interacted with miR-92-2-5p, which targeted genes participating in lactation and milk composition synthesis. Gga_circ_0003018, gga_circ_0003019 and gga_circ_0003020 could bind with let-7c-5p regulating SOCS3 in crop milk production. These findings provide the circRNAs expression profiles and facilitate the analysis of molecular mechanism of crop milk production in pigeon.

Differential expression of glucose metabolism-related genes and AMP-activated protein kinases in crop tissue of male and female pigeons (Columba livia domestica) during the incubation and chick-rearing periods.

The objective of this study was to explore the carbohydrate contents of crop milk, insulin and glucose concentrations in serum and the expression patterns of AMP-activated protein kinases (AMPKs) and genes related to glucose metabolism in pigeon crops during the breeding period. Crop milk was collected from squabs of rearing Day 1 (R1) to R6. Contents of total sugar and reducing sugar increased to the maximum levels at R6 (p < 0.05). Forty-two pairs of adult pigeons were allotted to seven groups by different breeding stages, and their crops and serum were sampled. No significant differences were found in either insulin or glucose levels in serum. The glucose transporter 2 gene level was the greatest at R15 in females, whereas it was at R7 in males. However, sodium-dependent glucose transporters 1 expression in both sexes decreased from incubation Day 17 (I17) to R7. In females, glucokinase expression peaked at R1, and at R1 and R7 in males. Pyruvate kinase mRNA levels peaked at R7 in females and at R15 males. The mRNA abundance of fructose-1,6-bisphosphatase 1 in both sexes and glucose-6-phosphatase in females decreased after I10. While phosphoenolpyruvate carboxykinase 1 expression decreased after I17 (p < 0.05). Protein levels of AMPKα in crops were minimized at R1 (p < 0.05). In females, expression of AMPKα1 and AMPKα2 was inhibited at I17 and R1 (p < 0.05). In males, AMPKα1 expression was decreased at R7 (p < 0.05) and AMPKα2 was reduced at I10 and R1. pAMPK expression was the lowest at I17 in females, and it was at R7 and R25 in males. Conclusively, glycolysis in pigeon crops was enhanced during chick-rearing, while gluconeogenesis was significantly inhibited. The stability of the insulin level suggests that it was probably not involved in the regulation of glucose metabolism in crop tissues.

Dynamic changes in protein concentrations of keratins in crop milk and related gene expression in pigeon crops during different incubation and chick rearing stages.

1. The objective of this study was to examine the keratin composition of crop milk, the variation of epithelial thickness and keratin (K) gene expression in samples from young pigeon during incubation and chick rearing.2. Crop milk was collected from 1-, 3- and 5-day-old squab crops for keratin content analysis. Results showed that K4 accounted for the highest proportion of all detected keratins.3. In total, 42 pairs of adult pigeons were allocated to seven groups according to different stages to collect crop samples. Gene expression studies showed that the K3 gene expression was maximised at rearing Day 15 (15) and R1 in males and females, respectively. K6a gene level was the greatest at R15 in females, whereas it peaked at incubation Day 4 (I4) in males. The K12, K13, K23 and K80 gene levels were inhibited at the peak period of crop milk formation in comparison with I4. In females, K cochleal expression peaked at I10, whereas it was the greatest at R25 in males. K4 and K14 gene expression was the highest at I10 in females, while K4 and K14 were minimised at I17 and R7 in males, respectively. Gene expressions of K5, K8, K19 and K20 in males and K19 in females were maximised at R1. The K5, K20 and K75 gene levels in females peaked at R7. K75 and K8 expressions in males and females reached a maximum value at R25 and I17, respectively.4. The epithelial thickness of male and female crops reached their greatest levels at R1 and had the highest correlation with K19.5. These results emphasised the importance of keratinisation in crop milk formation, and different keratins probably play various roles during this period. The K19 was probably a marker for pigeon crop epithelium development. The sex of the parent pigeon affected keratin gene expression profiles.

Lipid accumulation and oxidative stress in the crop tissues of male and female pigeons during incubation and chick-rearing periods.

The current study aimed to evaluate the changes in lipid accumulation and oxidative status in pigeon crops during different breeding stages. Forty-two pairs of adult pigeons were randomly assigned to 7 groups. Lipid droplet accumulation in pigeon crops was visualized by using oil red O staining from d 17 of incubation (I17) to d 7 of chick rearing (R7). Transmission electron microscopy analysis showed swollen mitochondria with disintegration of cristae and typical characteristics of endoplasmic reticulum stress in crop tissues at R1 compared with those at I4. During the peak of pigeon milk formation, the concentrations of reactive oxygen species, and oxidative damage markers (advanced oxidation protein products, 8-hydroxy-2 deoxyguanosine, and malondialdehyde) and the enzyme activities of total superoxide dismutase and glutathione peroxidase were all elevated significantly (P < 0.05). The protein concentration of B-cell lymphoma-2 associated X in crop tissues was significantly higher at R1, while the level of B-cell lymphoma-2 protein in males was the highest at I4 (P < 0.05). The ratio of B-cell lymphoma-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) in both male and female crops peaked at R1 (P < 0.05). Gene expression of the key enzymes involved in mitochondrial and peroxisomal fatty acid ß-oxidation was investigated in crops. In males, the gene expression of carnitine palmitoyltransferase 1a peaked at R15, and that of carnitine palmitoyltransferase 2 increased significantly from R1 to R15 (P < 0.05). The mRNA abundance of long chain 3-hydroxyacyl-CoA dehydrogenase increased to the maximum value at R1 and I17 in males and females, respectively. From I17 to R7, the mRNA levels of acyl-CoA oxidase 1 and acyl-CoA oxidase 2 were decreased in pigeon crops (P < 0.05). Conclusively, lipid droplet accumulation was found in male and female pigeon crops from the end of incubation to the early stage of chick rearing. Although antioxidant defence and mitochondrial fatty acid ß-oxidation were both mobilized, oxidative stress in crop tissues still occurred during the peak of milk formation.

Comprehensive Transcriptomic and Metabolomic Analysis Revealed the Functional Differences in Pigeon Lactation between Male and Female during the Reproductive Cycle.

Lactation is a unique reproductive behavior in pigeons, with the crop serving as the organ responsible for secreting pigeon milk. Both male and female pigeons can produce crop milk and rear their offspring through a division of labor. Since the time of the secretion of pigeon crop milk is different in the process of feeding the young, whether the metabolism and formation of pigeon milk use the same mechanism is a very interesting scientific question. However, the metabolic dynamics and underlying genetic mechanisms involved in the formation of pigeon crop milk remain unclear, particularly during the incubation-feeding reproductive cycle. In this study, we integrated lactation-associated metabolism and transcriptome data from the crop tissues of both male and female pigeons during the brooding and feeding stages. We mapped the changes in metabolites related to milk formation in the crop tissues during these stages. Through metabolome profiling, we identified 1413 metabolites among 18 crop tissues. During the breeding cycles, the concentrations of estrone, L-ergothioneine, and L-histidine exhibited the most dynamic changes in females. In contrast, estrone, L-anserine, 1-methylhistidine, homovanillate, oxidized glutathione, and reducing glutathione showed the most dynamic changes in males. Gender-specific differences were observed in the metabolome, with several metabolites significantly differing between males and females, many of which were correlated with cytokine binding, immunity, and cytochrome P450 activity. Using this dataset, we constructed complex regulatory networks, enabling us to identify important metabolites and key genes involved in regulating the formation of pigeon milk in male and female pigeons, respectively. Additionally, we investigated gender-associated differences in the crop metabolites of pigeons. Our study revealed differences in the modulation of pigeon crop milk metabolism between males and females and shed light on the potential functions of male and female pigeon milk in the growth, development, and immunity of young pigeons, an area that has not been previously explored. In conclusion, our results provide new insights into the metabolic regulation of pigeon crop milk formation during the brooding and breeding stages. Furthermore, our findings lay the foundation for the accurate development of artificial pigeon milk.

Effect of Akt activation on apoptosis-related gene expression in the crop tissues of male and female pigeons (Columba livia).

The current study investigated whether the expression of apoptosis genes in the pigeon crops was affected by the Akt signaling pathway during crop milk formation. First, 78 pairs of adult White King pigeons were randomly assigned to 7 groups, and the expression of apoptosis-related genes and Akt signaling pathway-related proteins in the crop tissues during different breeding stages were examined. The results showed that the mRNA levels of Bak, caspase-3, caspase-6, and caspase-9 in female crops all increased and reached their highest levels at d 17 of incubation (I17). In male crops, the levels of caspase-3 and caspase-9 gene expression peaked at d 1 of chick rearing (R1). The lowest level of Bcl-2 gene expression in females was observed at I17. The expression ratios of p-Akt (Ser473)/Akt and p-Akt (Thr308)/Akt in male crops decreased to their minimum at R1, while it was observed at d 7 of chick rearing (R7) in females. Second, 36 pairs of adult pigeons were divided into 3 groups and were subjected to SC79 injections with dosages of 0, 0.02, or 0.04 mg/kg bodyweight. The SC79 injections resulted in a considerable decrease in growth performance of pigeon squabs. In male crops, the expression ratios of p-Akt (Ser473)/Akt and p-Akt (Thr308)/Akt were significantly elevated in the 0.02 mg/kg SC79 group, while in female crops, they were higher in the 0.04 mg/kg SC79 group (P < 0.05). The SC79 injection inhibited the gene expression of Bak in female crops, but enhanced the gene expression of Bcl-2 in both male and female crops. In the 0.04 mg/kg SC79 group, a 50.7 to 75.7% decrease was observed in the expression of caspase-3, caspase-6, and caspase-9 in male and female pigeon crops. Expression of the caspase-8 gene and total Akt protein in pigeon crops was not changed in different breeding stages or after SC79 injection. In conclusion, the expression of genes related to mitochondria-dependent apoptosis can be regulated by the Akt signaling pathway, which may play a potential role in pigeon milk formation.

Targeted disruption of TORC1 retards young squab growth by inhibiting the synthesis of crop milk protein in breeding pigeon (Columba livia).

This study was conducted to explore the regulatory role of the target of rapamycin complex 1 (TORC1) signaling pathway in crop milk synthesis in breeding pigeons (Columba livia). Three groups of breeding pigeons in the lactation period (n = 30 pairs/group) were respectively injected with rapamycin (RAPA, a specific inhibitor of the target of rapamycin complex) at doses of 0 (vehicle, control), 0.6, or 1.2 mg/kg body weight (BW)/day via the wing vein for 7 days. The average daily feed intake (ADFI) and BW of the breeding pigeons and the BW of young squabs were respectively recorded throughout the experimental period. The breeding pigeons were sacrificed to collect their crop tissues, crop milk, and serum on the eighth day of the experiment. The results showed that neither 0.6 nor 1.2 mg/kg BW RAPA injection affected BW loss or ADFI in breeding pigeons (P > 0.05), while crop thickness and crop relative weight were significantly decreased (P < 0.05) in the 1.2 mg/kg BW rapamycin-injected group. Simultaneously, RAPA (especially at 1.2 mg/kg BW) decreased the crude protein, αs1-casein, αs2-casein, ß-casein, and amino acid contents (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Lys, His, Arg, and Pro) of crop milk (P < 0.05) and the concentrations of albumin, total protein, and uric acid in the serum of breeding pigeons (P < 0.05). Additionally, the expression of TORC1 pathway-related proteins (TORC1, S6K1, S6, 4EBP1, and eIF4E) was downregulated in the crop tissues of breeding pigeons by 0.6 or 1.2 mg/kg BW/day RAPA injection (P < 0.05). Accordingly, the average daily gain (ADG) of young squabs declined, and the mortality rate increased significantly (P < 0.05). Together, the results showed that RAPA reduced protein and amino acid levels in the crop milk of breeding pigeons and retarded young squab growth, suggesting a crucial role of TORC1 in crop milk synthesis in breeding pigeons.

The profiling of amino acids in crop milk and plasma and mRNA abundance of amino acid transporters and enzymes related to amino acid synthesis in the crop tissue of male and female pigeons during incubation and chick-rearing periods.

The present study was carried out to investigate the changes in amino acid (AA) contents of crop milk and plasma and mRNA abundance of AA transporters and AA synthesis-related enzymes in the crop tissue of male and female pigeons during incubation and chick-rearing periods. Forty-two pairs of adult White King pigeons with 2 fertile eggs per pair were randomly divided into 7 groups by different breeding stages. The AA content of crop milk decreased from day 1 (R1) to day 25 (R25) of chick rearing (P < 0.05). In both male and female adult pigeons, the contents of Thr, Leu, Val, His, Asp, and Pro in plasma increased to maximum levels on R25. Parental sex effect and interaction between stage and sex were observed in the AA contents of pigeon plasma (P < 0.05). For AA transporters, the mRNA abundances of SNAT2, ASCT1, LAT1, and y+LAT2 in the male crops reached the highest value on day 17 of incubation (I17), and the peak mRNA levels of PAT-1, xCT, b0,+AT, and CAT1 were found on R7 (P < 0.05). In females, the abundances of ASCT1, B0AT1, asc-1, and CAT1 mRNA peaked on R1, whereas the maximum levels of LAT1, PAT-1, b0,+AT, and y+LAT2 were observed on R7. For enzymes involved in AA synthesis, the highest gene expressions of glutamate dehydrogenase 1, acetolactate synthase in both parent pigeons, and L-threonine 3-dehydrogenase in female pigeon crops were attained on I17. The expressions of ornithine-δ-aminotransferase, glutamic-oxal(o)acetic transaminase 1, glutamic-oxal(o)acetic transaminase 2, asparagine synthetase, serine hydroxymethyltransferase 2, and glutamic-pyruvic transaminase 2 in both sexes and argininosuccinate lyase and L-threonine 3-dehydrogenase in males were the highest on R1. In conclusion, AA used for pigeon crop milk formation may originate from plasma and intracellular synthesis. The genes involved in AA transport and synthesis varied significantly with sexual effects, indicating that other factors should be considered in future explorations of the mechanism of protein formation in crop milk.

Identification of microRNA-Associated-ceRNA Networks Regulating Crop Milk Production in Pigeon (Columba livia).

Pigeon belongs to altrices. Squab cannot forage independently. Nutrition can only be obtained from crop milk secreted by male and female pigeon. miRNA could regulate many biological events. However, the roles of miRNA and ceRNA in regulating crop milk production are still unknown. In this study, we investigated the miRNAs expression profile of female pigeon crop, explored the potential key genes, and found the regulatory mechanisms of crop milk production. A total of 71 miRNAs were identified differentially expressed significantly. Meanwhile, miR-20b-5p, miR-146b-5p, miR-21-5p, and miR-26b-5p were found to be the key miRNAs regulating lactation. Target genes of these miRNAs participated mainly in cell development; protein and lipid synthesis; and ion signaling processes, such as cell-cell adhesion, epithelial cell morphogenesis, calcium signaling pathway, protein digestion, and absorption. In the ceRNA network, miR-193-5p was located in the central position, and miR-193-5p/CREBRF/LOC110355588, miR-460b-5p/GRHL2/MSTRG.132954, and miR-193-5p/PIK3CD/LOC110355588 regulatory axes were believed to affect lactation. Collectively, our findings enriched the miRNA expression profile of pigeon and provided novel insights into the microRNA-associated-ceRNA networks regulating crop milk production in pigeon.

Leucine promotes the growth of squabs by increasing crop milk protein synthesis through the TOR signaling pathway in the domestic pigeon (Columba livia).

Leucine (Leu) plays a critical regulatory role in protein synthesis, however, the effects and molecular mechanisms of Leu on crop milk protein in the domestic pigeons (Columba livia) are still unknown. Therefore, the study aimed to investigate the effects of dietary Leu supplementation on crop milk protein synthesis and the growth performance of squabs and the possible underlying mechanism. A total of 240 pairs of breeding pigeons (1102.3 ± 9.5 g/pair) were randomly assigned to 1 of 5 treatments, including a positive control (PC) diet that had adequate crude protein (crude protein, CP = 18%; Leu = 1.30%), a negative control (NC) diet that was low in CP (CP = 16%, Leu = 1.30%), and NC diets supplemented with Leu at 0.15%, 0.45%, or 1.05%. Compared with the NC diet, 0.15 to 0.45% Leu supplementation decreased BW loss and increased relative crop weight, crop thickness, and protein levels in the crop tissue and milk of breeding pigeons. However, dietary supplementation with 1.05% Leu inhibited ADFI in breeding pigeons. Dietary supplementation with 0.15 to 0.45% Leu decreased the mortality rate and increased the BW, eviscerated yield, and breast muscle yield of young squabs. The protein expression levels of the target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1), ribosomal protein S6 kinase (S6), eukaryotic initiation factor 4E binding protein 1 (4EBP1), and eukaryotic translation initiation factor 4E (eIF4E) were upregulated in the crop tissue of breeding pigeons in PC, 0.15% and 0.45% Leu-supplemented groups. Collectively, these results indicated that 0.15 to 0.45% Leu supplementation could decrease BW loss, increase milk protein synthesis in the crop of breeding pigeons, and enhance the survival rate and growth performance of young squabs through the TOR signaling pathway.

Differential expression of fatty acid transporters and fatty acid synthesis-related genes in crop tissues of male and female pigeons (Columba livia domestica) during incubation and chick rearing.

1. The growth performance of squabs reared solely by male or female parent pigeons was measured, and the changes of lipid content of crop milk and the expression profiles of genes potentially involved in lipid accumulation by crop tissues of parent pigeons were evaluated during incubation and chick rearing. 2. Squabs increased in body weight during 25 d of rearing, whereas both male and female pigeons lost weight after finishing rearing chicks, and the weight loss of male pigeons was significantly greater than that of female parent pigeons. Lipid content of crop milk from both parent pigeons gradually decreased to the crude fat level in the formulated diet after 10 d (R10) of chick rearing. 3. The gene expression of fatty acid translocase (FAT/CD36), fatty acid-binding protein 5 (EFABP) and acyl-CoA-binding protein (ACBP) in male pigeon crop tissue were the greatest at 17 d (I17) of incubation. In female pigeons, FAT/CD36 expression was the highest at I14, and both EFABP and ACBP expression peaked at I14 and R7. The expression of acetyl-CoA carboxylase and fatty acid synthase in male pigeons reached the maximum level at R1, while they peaked at I14 and I17, respectively in female pigeons. The gene expression of peroxisome proliferators-activated receptor-gamma (PPARγ) was the greatest at I17 in the male, while it was at I14 in the female. However, no regular changing pattern was found in PPARα gene expression in male pigeons. 4. These results indicated that male and female pigeons may make different contributions in rearing squabs. The gene expression study suggested that fatty acids used in lipid biosynthesis of crop milk probably originated from both exogenous supply and de novo synthesis. The sex of the parent pigeon affected the lipid content of crop milk and the expression profiles of genes involved in fatty acid transportation and lipogenesis.

Proceedings of the 2015 National Toxicology Program Satellite Symposium.

The 2015 Annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in Minneapolis, Minnesota, at the American College of Veterinary Pathologists/American Society for Veterinary Clinical Pathology/Society of Toxicologic Pathology combined meeting. The goal of this symposium is to present and discuss diagnostic pathology challenges or nomenclature issues. Because of the combined meeting, both laboratory and domestic animal cases were presented. This article presents summaries of the speakers' talks, including challenging diagnostic cases or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included hepatocellular lesions, a proposed harmonized diagnostic approach to rat cardiomyopathy, crop milk in a bird, avian feeding accoutrement, heat exchanger in a tuna, metastasis of a tobacco carcinogen-induced pulmonary carcinoma, neurocytoma in a rat, pituicytoma in a rat, rodent mammary gland whole mounts, dog and rat alveolar macrophage ultrastructure, dog and rat pulmonary phospholipidosis, alveolar macrophage aggregation in a dog, degenerating yeast in a cat liver aspirate, myeloid leukemia in lymph node aspirates from a dog, Trypanosoma cruzi in a dog, solanum toxicity in a cow, bovine astrovirus, malignant microglial tumor, and nomenclature challenges from the Special Senses International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Group.