Effects of early lactation body condition loss in dairy cows on serum lipid profiles and on oocyte and cumulus cell transcriptomes

Abstract

The objective of this study was to determine the effect of early lactation body condition (BC) loss in multiparous dairy cows on serum lipids and the effect of these changes on oocyte and cumulus cell transcriptomes. Body condition loss in dairy cattle after parturition is associated with reduced fertility and increased pregnancy loss. The complex interplay between BC, nutrition, dry matter intake, milk production, and time of calving has presented a barrier to understanding mechanisms leading to reduced fertility. We identified cows that lost BC (L group; n = 10) or maintained or gained BC (M/G group; n = 8) during the first 27 to 33 d in milk and investigated changes in serum fatty acids and oocyte and cumulus cell transcriptomes at 75 to 81 d in milk. The L group had increased serum levels of nonesterified fatty acids and mead acid, and reduced serum levels of petroselaidic acid and behenic acid. Transcriptome analyses revealed 38 differentially expressed genes (DEG) in oocytes and 71 DEG in cumulus cells of L (n = 3) compared with M/G group (n = 3). Network analysis connected serum fatty acid changes to downstream effects including reduced inflammatory response and mitochondrial membrane depolarization, increased production of reactive oxygen species, and functions related to fatty acid metabolism and cytoplasmic organization in oocytes. These effects were associated with predicted effects on signaling in oocytes through calcium, insulin, O-GlcNAcase (OGA), fibroblast growth factor receptor 4 (FGF4R), peroxisome proliferator activated receptor gamma coactivator 1 α (PPARGC1A), and phospholipase D2 (PLD2) pathways, with a connection to the cumulus cell via calcium signaling. These results connect BC loss following parturition to changes in serum lipid levels, and changes potentially affecting oocyte quality; thus, these results provide new insight into mechanism of reduced fertility.

Keywords: body condition loss; lipid metabolism; mitochondrial activity; oocyte transcriptome.

Figure 1.

Changes in BCS for multiparous dairy cows that either lost (n = 10) or maintained or gained (n = 8) body condition during the first 27 to 33 DIM. Body condition scores were evaluated weekly according to a 5-point scale with 0.1-point increments. The measurement in the final week of gestation was set as baseline. Body condition scores were again determined at 27 to 33 d after calving to determine change within scores. Circles denote baseline BCS, and squares denote postparturition BCS. Red symbols denote the animals employed for RNA sequencing. The numbers on the x-axis are animal numbers.

Figure 2.

Serum levels of nonesterified fatty acids (NEFA) for lactating dairy cows that either lost (n = 10) or maintained or gained (M/G; n = 8) body condition were assessed from blood collected at 76 to 82 DIM during the first 27 to 33 DIM. A significant difference between groups was set at P < 0.05 using Student’s t-test. Circles denote loss group animals, and squares denote M/G group animals. Red symbols are those employed for RNA sequencing.

Figure 3.

Principal component (PC) analysis of samples in RNA sequencing data set values after applying a variance stabilizing transformation within the DESeq2 package. Analysis indicates a very high degree of similarity between cows that lost (L) versus maintained or gained (M/G) body condition during the first 27 to 33 DIM.

Figure 4.

Volcano plots depicting comparisons of oocyte and cumulus cell transcriptomes from cows that lost (L; n = 3) versus maintained or gained (M/G; n = 3) body condition during the first 27 to 33 DIM. Figure contains panels for oocytes (left) and cumulus cells (right). The log₂(fold-change) comparing L versus M/G is along the x-axis, and the −log₁₀(FDR) along the y-axis, significance represented by horizontal dashed line at 1.3, equating to −log₁₀(FDR = 0.05). Shape denotes direction of change and significance: circle = nonsignificant difference; uptriangle = upregulated in L; and down-triangle = downregulated in M/G. FDR = false discovery rate.

Figure 5.

Effects of body condition loss on transcriptomes for lactating dairy cows that either lost or maintained or gained body condition during the first 27 to 33 DIM, as observed using Ingenuity Pathway Analysis (IPA). Canonical pathways, biological functions, and upstream regulators were identified by IPA as being significantly affected. Black closed circles denote IPA pathways, functions, or upstream regulators that are significantly affected. The red closed circle denotes an upstream regulator predicted to be activated. Open circles denote upstream regulators predicted to be affected but not expressed in the oocyte. Details of these effects are shown in Supplemental Table S3.

Figure 6.

Expanded network analysis linking fatty acids with significantly altered serum levels, upstream regulators, downstream differently expressed gene (DEG) targets, and their enrichment within pathways and functions. Figure consists of 3 boxed tiers: (1) Fatty acids significantly different between the loss (L) and maintain or gained (M/G) groups or with predicted activation/inhibition; (2) upstream regulators downstream of fatty acids and the DEG targets by said regulators; and (3) enriched canonical pathways (CP) and biological functions (BF). Factor not bounded by central dotted box (CALCA) denotes a cumulus cell DEG connecting to Ca²⁺ and insulin. Color fill of nodes denotes measured significant difference between L and M/G: red = increased in L; blue = decreased in L. Exterior color denotes Ingenuity Pathway Analysis (IPA) predicted z-score: red = activated; blue = inhibited; pink = positive trend not meeting significance. Regulators with bold font (OGA and TP53) denote those upstream regulators (UR) that were identified from IPA core analysis as significantly affected. Regulators with dashed red lines are those identified by the IPA Path Explorer tool to connect fatty acids to other regulators and DEG, but not initially indicated by the IPA core analysis. Gray lines indicate possible effects emanating from lipids that did not reach statistical significance for difference in serum concentrations. CP/BF membership of fatty acids, UR, and DEG denoted by superscript, corresponding to matched CP/BF superscript.

Figure 7.

Working model of how negative energy balance in lactating dairy cows ultimately affects oocyte quality and fertility. Lactation in animals in the loss group leads to a condition of negative energy balance and body condition (BC) loss with lipolysis. This changes serum lipid profiles, which in turn lead to transcriptome changes associated with possible meiosis defects, lipid toxicity, oxidative stress, and cellular damage, reducing oocyte quality and fertility.