The field study was conducted during a summer period with ambient temperatures above average for (Northern) German conditions. However, temperatures displayed a typical day-night cycle with higher values at daytime and lower temperatures during the night. In detail, temperature measurements revealed a mean ambient temperature of approx. 18.6 °C. Average minimum and maximum values were 15.7 °C and 21.7 °C, respectively. The temperatures within tents displayed a mean of 19.4 °C, with minimum and maximum mean values of 15.2 °C and 25.1 °C, respectively.

Body core (rectal) temperatures of all carcasses (n = 10) showed fluctuating temperatures in a circadian rhythm. They were slightly higher than ambient temperatures, especially during the first 2 days of the experiment. In addition, from 4 dpm onwards, open-field pigs displayed even higher rectal temperatures compared to environmental temperatures, most probably due to the heat produced by fly maggots, extensively colonizing the corpses (see below). Mean rectal temperature of the openly placed pigs was 25.3 °C during the experiment. Minimum and maximum rectal temperatures of open-field pigs were 18.7 °C and 24.9 °C, respectively. Tent pigs, by comparison, showed slightly lower temperatures than openly placed pigs with mean values of 19.7 °C and minimum and maximum temperatures of 17.2 °C and 23.3 °C, respectively (c.f. Fig. 1 and Supplementary Fig. S1).

Means of ambient (dark color) and rectal temperatures (light color) of open-field pigs (blue) and tents [46], including standard deviations. All mean temperature values vary between 15 and 25 °C. Rectal (body core) temperatures are higher in relation to respective ambient temperatures, especially rectal temperatures of open field cadavers

At the onset of the experiment, neither rigor, nor livor mortis was detectable in any of the animals. However, after that, significant differences were detected (Fig. 2).

Development of total body score (TBS) mean values of open-field pigs (blue squares) and tent pigs (red triangles), including standard deviations and trend lines. Both treatment groups depicted an increase of TBS values over the investigated time course of 10 dpm for both treatment groups. TBS values of open-field pigs are significantly higher than tent pigs

All open-field cadavers decomposed within only few days and went from a fresh stage to advanced decomposition very quickly (Fig. 2a). Total body scores developed from 3 at 0 dpm to 29 points within only 10 days (Fig. 3).

Representative progression of morphological changes during the decomposition of open-field pigs (top row) and tent pigs (bottom row) over the time course of 10 dpm

In detail, at the beginning of the experiment (0 dpm), all five pigs were in a fresh state. Shortly after death (approx. 2 h), adult flies started oviposition on the cadavers. Within the first 5 days of the experiment (5 dpm), all pigs showed typical signs of early decomposition, including extensive maggot and adult beetle colonization of head and trunk, green to black discoloration all over the cadaver. In addition, limbs and the abdominal of the cadavers were characterized by a marbling and maximum bloating, as well as extensive skin slippage.

Between 5 and 10 dpm, all open-field cadavers entered advanced decomposition stages, which was mainly characterized by extensive maggot colonization all over the cadaver, the beginning mummification of trunk and limbs, including leathery appearance of the skin and bone exposure. The trunks displayed dried brown areas with skin slippage and extensive hair loss. At 10 dpm, a complete bone exposure of the head and limbs was detectable and marked the onset of the skeletonization stage. In addition, some of the cadavers showed exposed bone in the abdominal region (mainly rib cage).

For a detailed description of morphological changes in the open-field pigs, see Supplementary File S1.

In general, the decomposition process of the pig cadavers placed inside tents was much slower than in the open-field pigs (Fig. 2b). Four of five pigs showed similar morphological changes at distinct PMI phases with total body scores from 3 at 0 dpm up to a maximum of 12 within 10 dpm. One pig showed an exceptionally advanced decomposition rate compared to the remaining cadavers with a TBS of 19 at 10 dpm (Fig. 3).

At the beginning of the experiment (0 dpm), all pigs were in a fresh state. At 5 dpm, tents were opened for the first time and revealed similar initial morphological changes amongst all cadavers, aligned with signs of early decomposition. These signs included green discoloration and bloating of the cadavers with maggot colonization (mainly in the mouth region) and marbling of the skin.

At 10 dpm, four pigs displayed a similar putrefaction progress, still typical for an early decomposition stage. Most prominent signs were bloating of head, neck, and limbs, post-bloated trunks, purging of decomposition fluids, skin slippage, and a green-to-black discoloration of the cadavers. One of the cadavers showed exceptional morphological changes and signs of an advanced decomposition phase. These changes included extensive skin slippage on neck and limbs, maggot colonization, and migration of post feeding larvae to the tent corners. In addition, a moist decomposition with foam building around the cadaver and bone exposure of trunk and limbs was detectable.

For a detailed description, see Supplementary File S2.

Due to the high temperatures and substantial insect activity, decomposition has progressed in a manner that muscle tissue collection was substantially hindered already after day 7 in the open-field pigs, whereas muscle samples from pigs in a sealed environment (tents) could still be obtained on day 10 postmortem in all animals. Results showed that investigated muscle proteins degraded in a similar fashion, regardless of exposure of the animals to the environment (open field or tent) and despite differences in the decomposition process and morphological changes amongst treatment groups. Even under extreme ambient temperatures, some proteins remained stable over the investigated time, while others displayed a complete degradation of the native form, in part accompanied by the occurrence of distinct degradation products at defined PMI phases.

Tropomyosin with its native double-band appearance remained stable over the time course of this field study in all open-field cadavers. The protein showed no signs of degradation, nor displayed any degradation products over the investigated time (Fig. 4A).

Results of the analysis of muscle protein degradation. a–e Representative Western blot results depicting muscle protein degradation patterns of tropomyosin (a), alpha tubulin (b), GAPDH (c), vinculin (d), and alpha actinin (e), over a time course of 7 dpm for open-field pigs, and over 10 dpm for tent pigs. f Heatmap of selected native proteins and degradation products, depicting the abundance of protein bands over the investigated time course. Native forms of alpha tubulin, GAPDH, vinculin and alpha actinin, as well as meta-vinculin showed a decrease of band intensity, the vinculin split product of 63 kDa (e) depicted an increase of band presence

In comparison, alpha tubulin and GAPDH fully degraded without the formation of any split products. In detail, the native band of alpha tubulin disappeared after 1 dpm in all open-field pigs (Figs. 4B and 5A). GAPDH fully degraded in 2 of 5 open-field animals over the investigated time (Figs. 4C and 5B), others displayed a fading in band intensity of the native protein.

Relative abundances of alpha tubulin (a), GAPDH (b), meta-vinculin (c), vinculin (d), vinculin degradation product 63 kDA (e), and alpha actinin (f), depicting the abundance of protein bands over the investigated time course of 7 and 10 dpm, respectively, for open-field pigs (blue line) and tent pigs (red triangle). Standard deviations are included and significances (p < 0.05) are depicted by asterisk in blue for open-field pigs and in red for tent pigs. All native proteins (a, b, d, f) as well as meta-vinculin (c) showed a decrease in relative abundance in both open-field and tent pigs. The vinculin degradation product of 63 kDa (e) depicted an increase in relative abundance over the investigated time in both treatment groups

The proteins vinculin and alpha actinin also exhibited a complete degradation of the native band over time in open field. This degradation was accompanied by the appearance of degradation products at distinct time points (Fig. 4D + E). Analysis showed that in openly placed animals, native vinculin was fully degraded after 3 dpm (Fig. 5D), and native alpha actinin completely disappeared at 7 dpm (Fig. 5F). Split products of alpha actinin with molecular weights between 80 and 55 kDa occurred from 3 dpm onwards but displayed no statistical regularity in their appearance, whereas some splice variants and degradation products of vinculin appeared at specific time points with high probability. Specifically, the vinculin splice variant with a molecular weight of 135 kDa, often referred to as meta-vinculin, degraded within the first 3 days postmortem (Fig. 5C). In addition, the vinculin protein band with a molecular weight of approx. 63 kDa significantly appeared from 3 dpm onward (Fig. 5E).

Like in open-field pigs, native tropomyosin remained stable over the investigated time course in all tent pigs without any signs of degradation (Fig. 4A).

Western blot results revealed significant loss of the native alpha-tubulin band in all tent carcasses on sampling day 5 dpm (p < 0.05), indicating a full degradation of the protein within the first 5 days postmortem (Figs. 4B and 5A).

Similar to open-field pigs, 4 of 5 tent pigs showed a complete degradation of native GAPDH at 10 dpm (Figs. 4C and 5B). The remaining pig showed a stable native protein band with a fading of band intensity over the investigated time.

Tent cadavers also showed similar degradation patterns of vinculin and alpha actinin compared to open field pigs (Fig. 4D + E). The native forms of the proteins in tent pigs were fully degraded at 5 dpm for vinculin and within 5 to 10 dpm for alpha actinin (Fig. 5D + F). The meta-vinculin splice variant was fully degraded in all tent carcasses at 5 dpm (Fig. 5C), indicating a full degradation of the protein band within this time period. The vinculin split product of 63 kDa appeared in tent pigs from 5 dpm onwards (Fig. 5E). As in open-field pigs, tent carcasses also displayed degradation products of alpha actinin but showed no statistical regularity in their appearances.