Elsevier

Theriogenology

Volume 84, Issue 8, November 2015, Pages 1350-1361.e6
Theriogenology

Research article
The effect of energy balance on the transcriptome of bovine granulosa cells at 60 days postpartum

https://doi.org/10.1016/j.theriogenology.2015.07.015Get rights and content

Abstract

Dairy cows expend great amounts of energy during the lactation peak to cope with milk production. A state of negative energy balance (NEB) was suggested as a cause for the suboptimal fertility observed during this period, via an interaction with ovarian function. The objective of this study was to identify the impact of NEB on gene expression in granulosa cells of dairy cows at 60 days postpartum and to suggest a potential treatment to improve ovarian function. Dairy cows at 60 days postpartum from 10 typical medium-sized farms were synchronized using a single injection of prostaglandin. Dominant follicles  were collected 42 hours later by transvaginal aspiration. Blood concentrations of beta-hydroxybutyrate (BHB) on the day of aspiration were used to classify animals into two groups: severe NEB (high BHB, n = 12) and mild NEB (low BHB, n = 12). The transcriptomes of granulosa cells from both groups were contrasted using microarrays, and the differentially expressed genes were analyzed using Ingenuity Pathway Analysis to identify affected functions and potential upstream regulators. Genes linked with cellular organization (KRT4 and PPL), proliferation (TACSTD2), and fatty acids metabolism (VNN2) were downregulated in granulosa cells from animals with severe NEB. Several genes linked to decitabine, a hypomethylating agent, and with beta-estradiol, were downregulated in the severe NEB group. Numerous genes linked to vitamins A and D were also downregulated in this group of cows, suggesting a potential deficiency of these vitamins in dairy cows during the postpartum period. This study supports the idea that energy balance has an impact on follicular dynamics which could be detrimental to resumption of fertility after calving.

Introduction

Dairy production is an important aspect of the food industry in many countries. As the pregnancy and birth of a calf are essential to the onset of milk production in the cow, the dairy industry requires fertile animals. However, the past decades have seen a noticeable drop in the fertility of Canadian dairy cows, mainly for the Holstein breed. This trend is worrisome for producers, as it is associated with additional expenses and lower profits. The main problems observed are longer intervals between calving and first insemination [1], decreased conception rates [2], abnormal estrus [3], and increased pregnancy losses [4]. These problems considerably reduce the longevity of dairy cow herds: In 2013, 8.2% of dairy cows registered in Canada were culled for reproductive reasons (Canadian Dairy Information Centre). No single factor was proven to be the cause of the declining fertility in dairy cattle, but increased lactation demands was suggested to play a major role. Global conception rates have increased during the past few years, and the milk yield per animal also increased over the same period [5]. However, although previous studies highlighted relations between milk production and several aspects of cattle reproduction, they were not able to report a direct relationship between milk yield and fertility [6], [7].

During the postpartum period, dairy cows experience a period of negative energy balance (NEB) and need to mobilize their body reserves to cope with the energy demand of lactation [8]. The amount of energy required for milk production increases quickly after calving, whereas the increase in dry matter intake lags behind [9]. Animals selected for milk production tend to partition more energy toward milk rather than restoring body reserves [10], [11] and are therefore more susceptible to experience a prolonged period of NEB. Fertility in heifers and beef cows has not declined in a similar way as fertility in lactating cows [12], [13], [14], supporting the hypothesis of an excessive metabolic stress during lactation. However, energy balance status may affect reproduction [15] more than milk yield, as it induces metabolic stress.

During periods of fasting or of high energy demand such as lactation, body energy reserves are mobilized and lipid metabolism is activated. The released fatty acids are oxidized by the liver to produce ketone bodies, which can transport energy from the liver to other tissues. The concentration of these molecules in blood serum increases with lipid mobilization. The most abundant ketone body is beta-hydroxybutyrate (BHB), and its blood concentration is an indicator of ketosis in cattle, with subclinical ketosis ranging from 1200 to 1400 μmol/L [16]. Cows with a higher body condition score (BCS) at calving are more likely to experience ketosis [17] because they eat less than thin cows and will therefore need to mobilize more body reserves to cope with the energy demand of lactation [18], [19]. In dairy cows, high concentrations of BHB were linked to periparturient diseases [20].

The follicular environment greatly influences the competence of the oocyte [21] and, therefore, ultimately contributes to embryo quality. Granulosa cells' transcriptome is known to be altered by numerous aspects of follicular development, including growth stage [22], [23]. In vitro studies have shown that some blood metabolites are linked to energy balance, such as nonesterified fatty acids and BHB, and can influence bovine granulosa cell functions [24], [25], [26]. Our hypothesis is that energy balance has an impact on bovine granulosa cells' transcriptome. The objective of this study was to analyze the transcriptomic profiles of granulosa cells from dairy cows with more or less severe NEB at 60 days postpartum, to identify key genes and pathways connecting energy balance and follicular development.

Section snippets

Animals

Lactating Holstein cows were selected from 10 medium-sized dairy farms with 75 to 300 animals. Several small farms, with dietary and management differences, were preferred to a single large farm as a way to allow broader application of the conclusions. Animal diets and management of the farms were taken into consideration, and animals were paired accordingly for all contrasts. All samples were collected during the winter of 2013 to 2014. The farm managers were asked to provide milk recording

Microarray data

The data presented in this publication have been deposited in NCBI's Gene Expression Omnibus [35] and are accessible through GEO Series accession numbers GSE66116. The HCA graph (Supplementary Figure 1) revealed an absence of complete segregation between the groups. The BHB profile did not seem to have a similar effect in every animal and led to poor segregation. After normalization, a cutoff set at a symmetrical fold change of 1.5, an intensity of 8, and a P value of 0.05 was applied and it

Microarrays

This study used transcriptomic profile analysis of granulosa cells from dairy cows to identify genes and pathways connecting energy balance and follicular development, to provide suggestions to improve fertility in cattle. Animals were pooled to increase the number of animals per group (12 cows instead of four, as there are four arrays per slide). Pools reduce individual variations and background noise, but they also decrease the sensitivity of the analysis. However, the goal of this study was

Acknowledgments

The authors acknowledge Jean-Michel Guay and his assistants for the collection of samples. Annie Girard was supported by MAPAQ (Ministère de l'agriculture, des pêcheries et de l'alimentation du Québec), whereas Marc-André Sirard and Isabelle Dufort by NSERC (Natural Sciences and Engineering Research Council of Canada).

Author contributions: Annie Girard and Marc-André Sirard conceived the study and wrote the article. Annie Girard supervised sample collections, performed the extractions,

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