In this study, a total of 678 mitochondria were measured from cow adipocytes using transmission electron microscopy (TEM) images (Fig. 1a–f). Mitochondrial dimensions were assessed, with the length (perpendicular to the cristae) referred to as “height” and the thickness (parallel to the cristae) referred to as “width” (Fig. 2j). The overall mean length of mitochondria across all samples was 0.64 μm (SD ± 0.414), and the mean thickness was 0.25 μm (SD ± 0.089). The maximum recorded length of a mitochondrion was 3.79 μm, while the maximum thickness was 1.13 μm. Conversely, the minimum length and thickness observed were 0.11 μm and 0.10 μm, respectively. These measurements suggest a broad range of mitochondrial sizes, highlighting variability in mitochondrial morphology within adipocytes. A notable observation from the mitochondrial size distribution was the existence of a consistent “exclusion zone” — a specific range of size combinations (approximately 0.7–1.2 μm in length and 0.4–0.6 μm in thickness) that were systematically underrepresented across all animals and groups (Fig. 2d–i). Given that all animals were sampled under identical protocols and conditions, and the absence occurred across both adipose depots and BCS groups, it is unlikely that this pattern resulted from the timing of collection alone. Instead, the forbidden zone may reflect intrinsic biophysical or structural constraints on mitochondrial morphology in bovine adipocytes.
Representative examples of adipocyte mitochondria in cows. (a) Adipocyte mitochondria examples from the perirenal region of the cachectic group (TEM, scale 0.5 μm). (b) Adipocyte mitochondria examples from subcutaneous flank region of the cachectic group are shown below panel (a) (TEM, scale 0.5 μm). (c) Adipocyte mitochondria examples from subcutaneous flank region of the normal weight group (TEM, scale 1 μm), and (d) below this panel are some examples of mitochondria from the perirenal region of the normal weight group (TEM, scale 0.5 μm). (e) Adipocyte mitochondria examples in the overweight group. The first panel shows examples of mitochondria in the subcutaneous flank region (TEM, scale 0.5 μm), whereas (f) the panel below the first shows mitochondria from the perirenal region (TEM, scale 2 μm). The white arrows point to the mitochondria in the picture. Please note that the magnification of the image areas in panels (b) and (f) is only a close-up for detailed inspection. The anatomical location where the adipose tissue was sampled is represented on the cow drawings, where the triangle shows the perirenal region (B) and the star indicates the subcutaneous flank region (A). In order to represent the classification process, the Body Condition Score (BCS) scale and the group range (G) on that scale are positioned in front of each cow drawing. Note: In transmission electron microscopy, lipid droplets appear as large, electron-lucent (light) areas occupying most of the adipocyte volume. The cellular cytoplasm and mitochondria are confined to the peripheral rim. These “empty” regions correspond to the internal space of the lipid droplet. Also, the micrographs were selected for image clarity and structural detail, not for direct size comparison. All morphometric analyses were conducted using calibrated image analysis software, which accounts for magnification differences automatically. Panels (a) and (b) are illustrative examples of mitochondrial morphology and are not meant for visual size comparison. All measurements reported in the Results were performed using calibrated image datasets and are independent of figure display scale.
Distribution of mitochondria in the adipose tissue from cows. (a) Distribution of mitochondrial length and thickness (µm) in cow adipocytes from the perspective of anatomical regions. (b) Distribution of mitochondria volume in adipocytes from cows. The line with rhombus markers represents the volume of mitochondria in adipocytes from subcutaneous flank region for each of the three groups (x-axis). The line with square markers represents the volume of mitochondria in adipocytes in the perirenal region, also for each of the three groups (x-axis). The y-axis shows the volume of mitochondria (µm3). The dotted line represents the average of the two anatomical areas for each of the three groups. (c) Shows the distribution of mitochondrial length and thickness (µm) in adipocytes from cows, from the perspective of the groups. (d–f) General distribution of 678 mitochondria in adipocytes from cows. The lengths of mitochondria are shown on the x-axis (µm) and the thickness is given on the y-axis (µm). In panel (f) a rhombic shape can be observed which indicates the region of missing dimensions. (g–i) Shows the contour lines indicating the density of points in panels d, e and f. (j) The meaning of measurements. A dashed line with arrows in both directions shows the length of mitochondria and the thickness is indicated by the solid line with arrows in both directions. Notice that the length is considered as a perpendicular to the cristae and the thickness is taken parallel to the cristae. In panel (a), a statistically significant difference was observed in mitochondrial length between cachectic and normal-weight cows (p = 0.012), indicated by a horizontal bracket.
When plotted, all adipocyte mitochondria lengths fall within the range of 0.7–1.2 μm, while the thicknesses are within 0.4 μm to 0.6 μm (Fig. 2d–i). This finding suggests potential structural constraints that limit mitochondrial morphology within specific size ranges, possibly influenced by cellular architecture or lipid droplet interactions within adipocytes.
Mitochondrial characteristics by body condition score (BCS) groups
Our study investigated the mitochondrial size dynamics within three BCS categories: cachectic, normal weight, and overweight. Mitochondria were measured in two distinct anatomical regions: the subcutaneous flank area (A) and the perirenal area (B). These regions were selected to explore potential regional differences in mitochondrial morphology in relation to body condition. Each BCS group included three animals, one per anatomical region, with individual identifiers and sample weights provided in the supplementary tables below.
Cachectic group
In the cachectic group, which represents cows with a low BCS, mitochondria from both the subcutaneous flank area (A) and the perirenal area (B) were analyzed (Fig. 1a). The average mitochondrial length in the subcutaneous area (A) was 0.475 μm (± 0.035), and the average thickness was 0.275 μm (± 0.063). In the perirenal area (B), the average length was slightly lower at 0.46 μm (± 0.098), and the thickness was 0.22 μm (± 0.042) (Table 1).
Normal weight group
For cows in the normal weight group, mitochondria in the subcutaneous area of the flank (A) had an average length of 0.675 μm (± 0.077) and an average thickness of 0.245 μm (± 0.021) (Fig. 1c). In the perirenal area (B), mitochondria were shorter on average, with a length of 0.475 μm (± 0.049), but exhibited a greater thickness of 0.315 μm (± 0.063) compared to the subcutaneous region. The calculated average volume of mitochondria was 0.0318 μm3 in the subcutaneous area (A) and 0.0369 μm3 in the perirenal area (B) (Table 1).
Overweight group
In the overweight group, representing cows with a high BCS, mitochondrial dimensions were also assessed in both regions (Fig. 1e). In the subcutaneous flank area (A), the average mitochondrial length was 0.675 μm (± 0.176), while the thickness was 0.23 μm (± 0.0141). Mitochondria in the perirenal area (B) had a slightly longer average length of 0.58 μm (± 0.1979) and a thickness of 0.315 μm (± 0.063). The average mitochondrial volume in the subcutaneous area (A) was 0.028 μm3, which was lower than the volume in the perirenal area (B) at 0.045 μm3 (Table 1).
Distribution and correlations of mitochondrial sizes
The distribution of mitochondrial sizes across the different BCS groups reveals varying trends in different anatomical regions (Fig. 2a–c). In general, the mean length and thickness of mitochondria in adipocytes from the subcutaneous flank area (A) across all groups were 0.608 μm (± 0.115) and 0.25 μm (± 0.0229), respectively. In the perirenal area (B), the average length was 0.505 μm (± 0.0653) and the average thickness was 0.283 μm (± 0.0548) (Fig. 2a,c). The volume of mitochondria showed a linear increase from the cachectic to the overweight groups, but this trend differed between anatomical regions (Fig. 2b). The subcutaneous area (A) displayed relatively stable mitochondrial volumes across groups, with no statistically significant differences between cachectic and overweight cows (p > 0.05). In contrast, the perirenal area (B) exhibited similar volumes for the cachectic and overweight groups, with the highest volume observed in the normal weight group. The maximum average mitochondrial volume was 0.045 μm3 in the perirenal area (B) of the overweight group, while the minimum was 0.017 μm3 in the same area of the cachectic group. Interestingly, while the subcutaneous area (A) showed higher volumes in the normal weight group compared to the overweight group, the reverse was true for the perirenal area (B). Patterns of mitochondrial volume and morphology may differ depending on both anatomical location and body condition, potentially influenced by region-specific metabolic needs or biochemical signals (Fig. 2b).
Analysis of mitochondrial morphological adaptations
Our study also highlights that mitochondria in different anatomical areas may preferentially adapt their morphology based on adipose tissue expansion. For example, in the normal and overweight groups, mitochondria in the subcutaneous region (A) maintained a consistent length (~ 0.675 µm) but showed differences in thickness (~ 0.25 µm vs. 0.23 µm) (Table 1). Conversely, mitochondria in the perirenal area (B) demonstrated more variability in length rather than thickness, indicating potentially different adaptive responses or roles in these regions. Plots of mitochondrial dimensions (length vs. thickness) for both regions confirm a non-uniform distribution and the presence of an “exclusion zone” where certain sizes are absent (Fig. 2d–f). This zone might be related to structural limitations imposed by lipid droplets or other intracellular components in adipocytes. Contour lines further illustrate the density and prevalence of specific mitochondrial sizes commonly observed in cow adipose tissue (Fig. 2g–i).
These findings indicate significant variability in mitochondrial size and distribution in cow adipocytes, influenced by both body condition and anatomical location. Our study suggests that mitochondria adapt their morphology and size dynamically in response to metabolic demands associated with different body conditions and fat depots. Future biochemical analyses are warranted to elucidate the underlying mechanisms driving these morphological adaptations and their implications for metabolic health and disease. The data presented in Table 1 highlight the differences in mitochondrial length, thickness, and volume across body condition scores (BCS) groups (cachectic, normal weight, overweight) and anatomical regions (subcutaneous area flank (A) and perirenal area (B)). In the subcutaneous area (A), the average mitochondrial length increases from the cachectic group (0.475 ± 0.04 μm) to the normal weight group (0.675 ± 0.08 μm) and remains stable in the overweight group (0.675 ± 0.18 μm). However, in the perirenal area (B), there is a notable increase in mitochondrial length from the cachectic group (0.460 ± 0.10 μm) to the overweight group (0.580 ± 0.20 μm), suggesting region-specific dynamics in mitochondrial adaptation as BCS increases. The mitochondrial thickness in the subcutaneous area decreases slightly from the cachectic (0.275 ± 0.063 μm) to the overweight group (0.230 ± 0.014 μm), while in the perirenal area, thickness shows a marked increase from cachectic (0.220 ± 0.04 μm) to normal weight (0.315 ± 0.06 μm) but remains constant from normal weight to overweight.
These trends suggest differential adaptation mechanisms between fat depots. Mitochondrial volume also shows distinctive patterns between anatomical regions. In the subcutaneous area (A), the mitochondrial volume remains relatively consistent across the BCS groups (0.0281 to 0.0318 μm3), while in the perirenal area (B), it significantly increases from cachectic (0.0174 μm3) to overweight (0.0451 μm3). This indicates that mitochondria in the perirenal fat depot may undergo greater morphological enlargement as BCS increases, compared to those in subcutaneous fat. The coefficient of variation (CV%) indicates that there is greater variability in mitochondrial size measurements within the overweight group, particularly for length (CVLA = 26.1%, CVLB = 34.5%). This variability may reflect a more heterogeneous population of mitochondria adapting to shifting physiological states in overweight cows. While stage of lactation and associated energy demands may contribute to these patterns, the increased variability was observed primarily in the perirenal depot of overweight animals — a group that included cows in mid, late, and post-calving stages. This suggests that the observed mitochondrial heterogeneity likely results from both local metabolic adaptations and broader energy management strategies. We note that although mitochondrial length can in some cases be influenced by fission or fusion dynamics, the probability of capturing such transient events in fixed tissue sections is low, and our measurements reflect static morphological states as observed in transmission electron micrographs. Future studies with tighter lactation-stage controls are warranted to disentangle these overlapping factors. In other words, one inportant observation is the greater variability in mitochondrial size (length and volume) within the overweight group, particularly in the perirenal fat depot. This increased variability, as indicated by the higher coefficient of variation (CV%), suggests a more heterogeneous population of mitochondria in this group. While speculative, this heterogeneity may be consistent with diverse structural states that warrant further investigation into possible links with metabolic demand or cellular stress. However, functional conclusions would require additional molecular or biochemical validation. Please note that Fig. 2 complements Table 1 by visually illustrating the distribution and variability of mitochondrial dimensions across different BCS groups and anatomical regions, providing an intuitive understanding of the data trends that may not be immediately apparent from the table alone. Therefore, this approach allows for a more nuanced interpretation of mitochondrial size dynamics.
Statistical analysis of mitochondrial dimensions
The statistical analysis was performed to evaluate the differences in mitochondrial length, thickness, and volume across the three Body Condition Score (BCS) groups: cachectic, normal weight, and overweight. An analysis of variance (ANOVA) was conducted to test for significant differences between groups, followed by Tukey’s Honest Significant Difference (HSD) post-hoc test for pairwise comparisons. All statistical analyses were performed using standard software, with a significance level set at p < 0.05. The results of the ANOVA revealed significant differences in mitochondrial length across the three BCS groups (F(2,15) = 12.8, p = 0.001). Post-hoc tests showed that cachectic cows had significantly smaller mitochondria compared to both normal weight and overweight cows (p < 0.01), while no significant difference in length was observed between normal weight and overweight cows (p > 0.05). The 95% confidence intervals (CI) for mitochondrial length ranged from 0.445 to 0.505 μm in the cachectic group, 0.620 to 0.730 μm in the normal weight group, and 0.585 to 0.765 μm in the overweight group, confirming distinct mitochondrial size differences between cachectic and other cows. Similarly, mitochondrial thickness differed significantly between the groups (F(2,15) = 9.2, p = 0.015), with the cachectic group exhibiting thinner mitochondria compared to the normal weight and overweight groups (p < 0.05). The 95% confidence intervals for thickness ranged from 0.250 to 0.300 μm in the cachectic group, 0.230 to 0.260 μm in the normal weight group, and 0.220 to 0.240 μm in the overweight group. Mitochondrial volume also varied significantly between anatomical regions and body condition scores (F(2,15) = 8.6, p = 0.010). Notably, mitochondrial volumes were smaller in the perirenal area of cachectic cows compared to the normal and overweight groups (p < 0.01), while no significant differences in volume were found between the normal and overweight groups in the subcutaneous area (p > 0.05). The cachectic group had the lowest mitochondrial volume, with a mean of 0.0174 μm3 in the perirenal area and 0.0281 μm3 in the subcutaneous area. These results demonstrate that mitochondrial length, thickness, and volume are significantly influenced by body condition and anatomical location (p < 0.05), with the most pronounced and statistically significant reductions observed in cachectic cows, particularly in the perirenal depot. The statistical significance of these findings, supported by p-values and confidence intervals, suggests that mitochondrial morphology is a crucial factor in the metabolic adaptations of dairy cows across different body conditions.