Fig 1.
Pharmacological protocol, treatment conditions, and timing of ultrasound guided transvaginal follicular aspiration of the ovulatory follicle in lactating dairy cows maintained at thermoneutral or heat stress conditions [20]. At time of transport (35 h after [ha] final PGF2α), cows were randomly allocated to either thermoneutral or heat stress conditions. For thermoneutral conditions, cows were maintained at ~67 temperature-humidity index (THI). Heat stress conditions consisted of THI being steadily increased (final THI ~83) starting within 2 h of final GnRH analog administration. After ~12 h exposure to elevated THI, heat-stressed cows were suddenly cooled. Contents of the dominant follicle (DF) were aspirated ~16 ha GnRH. Ultrasonography (US) and blood sampling (BS) were done to monitor follicle turnover and growth as well as circulating hormone levels (estradiol, progesterone and LH). CIDR = progesterone containing controlled internal drug releasing device.
Table 1.
Characteristics of cows exposed to thermoneutral or heat-stress conditions.
Table 2.
Detection limits and precision of custom bovine 15-plex cytokine panel.
Table 3.
Characteristics of the periovulatory follicle and aspirated fluid.
Fig 2.
Characteristics of peptides identified within follicular fluid.
The distribution of percent of sequence coverage by identified peptides (A), number of peptide sequences unique to a protein (B), calculated molecular weight (C) and theoretically calculated isoelectric point (D) for the 339 proteins identified in follicular fluid.
Fig 3.
Gene ontology classification for the proteins identified in follicular fluid.
Cellular component (A), biological processes (B) and molecular function (C).
Fig 4.
Principal component analyses and hierarchical clustering of proteins identified in follicular fluid.
Principal component (PC) analysis plot (A) using singular value decomposition to calculate variance in the follicular fluid (FF) proteome collected at ~16 h post GnRH from thermoneutral (exposed to thermoneutral conditions; TN) and hyperthermic (exposed to heat stress conditions; HS) cows. The data points refer to FF samples from individual cows with temperature conditions identified by colors in legend. Prediction ellipses indicate 95% confidence region for each temperature group. Hierarchical clustering heat map (B) depicts abundance pattern of the 339 proteins identified. The heat map indicates high (dark red), low (grey) and intermediate (white) abundances for individual proteins (rows). The columns represent FF samples from individual cows with temperature identified at top of column by colors in legend.
Table 4.
Differentially-abundant proteins in follicular fluid from hyperthermic versus thermoneutral cows.
Table 5.
Functional annotation clustering of proteins identified to be altered in follicular fluid due to hyperthermia.
Table 6.
Reactome pathways over-represented in proteins identified to be altered in follicular fluid due to hyperthermia.
Fig 5.
Levels of bradykinin and transferrin within follicular fluid from thermoneutral, intermediate and hyperthermic cows.
Intrafollicular levels of bradykinin (pg per mg total protein; Panel A) and transferrin (μg per mg total protein; Panel B). ABBars (least squares means ± SEM) that do not share a letter differ significantly (P ≤ 0.05).
Fig 6.
Circulating levels of bradykinin and transferrin within thermoneutral, intermediate and hyperthermic cows.
Levels of bradykinin (A) and transferrin (B) in serum collected at GnRH administration but before environmental treatments were applied to cows and at time of dominant follicle (DF) aspiration (~16 h after GnRH administration).
Table 7.
Levels of cytokines within follicular fluid aspirates.
Table 8.
Levels of circulating cytokines.
Fig 7.
Simplified depiction of hyperthermia impacts on the intrafollicular complement and coagulation cascade.
The effects of hyperthermia on protein levels within follicular fluid designated with color; red for greater than 2-fold increase in abundance, pink for moderate increases (1.2 to 1.7-fold) in abundance, and blue for decreases (1.6 to 2-fold) in abundance. Bradykinin peptide is released from precursor kininogen through proteolysis mediated by kallikrein enzyme [45]. The proinflammatory actions of bradykinin are inhibited by plasma metalloproteases carboxypeptidase N catalytic chain 1 (CPN1; [46]) and carboxypeptidase B2 (CPB2; [47]). Kininogen can also serve as a substrate for cathepsin B or mannan binding lectin serine peptidase 2 (MASP2), though proteolysis with either enzyme does not release bradykinin [48, 49]. In addition, kininogen can function as a weak inhibitor of cathepsin B [49]. Alpha-2-Macroglobulin (A2M) is an anti-proteinase that complexes with proteinases such as kallikrein [50] to inhibit proteolytic activity.