Elsevier

Animal Reproduction Science

Volume 167, April 2016, Pages 40-50
Animal Reproduction Science

Intracellular lipid dysregulation interferes with leukocyte function in the ovaries of meat-type hens under unrestricted feed intake

https://doi.org/10.1016/j.anireprosci.2016.02.002Get rights and content

Highlights

  • Leukocytes play an active role in the ovulatory processes of meat-type Red Feather country chickens that are disturbed by unrestricted feeding.

  • Impaired leukocyte and granulosa cell interactions in Red Feather hens fed ad libitum results in tissue inflammation and delayed ovulatory processes.

  • Palmitic acid, increased ceramide formation and reactive oxygen species appear operative in the mechanisms that result in perturbation of cytokine and matrix metalloprotease activities of granulosa cells, heterophils and monocytes.

Abstract

Meat-type Red-feather country hens fed ad libitum (AD-hens) exhibit obesity-associated morbidities and a number of ovarian irregularities. Leukocyte participations in ovarian activities are unstudied in AD-hens. In contrast to feed-restricted hens (R-hens), ovulatory process of the F1 follicle appeared delayed in AD-hens in association with reduced F1 follicle progesterone content, gelatinase A (MMP-2) and collagenase-3 (MMP-13) activities coincident with elevated IL-1β and no production (P < 0.05), and increased leukocyte infiltration of inflamed necrotic follicle walls. Extracts of AD-hen F1 follicle walls induced greater leukocyte migration than extracts from F1 follicle wall extracts of R-hens (P < 0.05). Co-cultures of granulosa cells with increasing numbers of leukocytes from either AD-hens or R-hens exhibited dose dependent reductions in progesterone production and increases in cell death. AD-hen leukocytes were less proapoptotic than their R counterparts (P < 0.05). Granulosa MMP-13 and MMP-2 activities were also suppressed in the co-cultures with heterophils or monocytes in a dose-dependent manner (P < 0.05). AD heterophils and R monocytes had a greater inhibitory effect on MMP activities in the co-cultures than their respective counterparts (P < 0.05). Both basal and LPS-induced IL-1β secretion and MMP-22 or MMP-2 activities in freshly isolated AD-hen leukocytes were reduced (P < 0.05). Exposure of AD or R leukocytes to 0.5 mM palmitate impaired IL-1β secretion and MMP-22 or MMP-2 activity. Inhibition of ceramide synthesis with FB1 and ROS production with n-MPG scavenging rescued MMP activity and IL-1β production in palmitate treated heterophils, but exacerbated monocyte suppression. These latter findings suggest that intracellular lipid dysregulation in leukocytes contributes to ovarian dysfunction in AD-hens.

Introduction

Egg production is impaired in several strains of meat-type hens when provided with ad libitum access to feed (AD-hens) (Hocking et al., 1989, Yu and Robinson, 1992, Pan et al., 2014). Poultry meat from country chickens is preferred in many Asian markets and interestingly Red Feather hens of meat-type country chickens manifest dyslipidemia and systemic inflammation in association with impaired egg production as occurs in commercial meat-type hens derived from Cornish x Plymouth Rock broiler strains (Chen et al., 2006, Pan et al., 2012, Pan et al., 2014) In both cases, systemic inflammation was due to a condition termed lipotoxicity (Unger, 2002, Prieur et al., 2010) that develops when cellular fatty acid availability exceeds cellular ability to dispose of fatty acids via β-oxidation or store them as neutral lipids. Under these conditions, cells dysfunction can undergo apoptosis and further induce chronic in situ inflammation. Cellular lipid dysregulation was evident in lipotoxic broiler AD-hens as elevated tissue saturated fatty acid concentrations and ceramide accumulation (Chen et al., 2006, Pan et al., 2012, Xie et al., 2012). Changes in tissue lipid composition were related to pronounced granulosa cell apoptosis, increased follicle interleukin-1β (IL-1β) production and leukocyte infiltration in the prevoulatory F1 follicle (Pan et al., 2012, Pan et al., 2014, Xie et al., 2012, Walzem and Chen, 2014). However, effects within individual cell types were not studied.

In mammals, it is well documented that leukocytes contribute to the tissue remodeling during the ovulatory process (Brannstrom and Enskog, 2002, Bukulmez and Arici, 2000). Activation of leukocytes recruited into the follicles and following release of regulatory factors such as inflammatory cytokines, proteases, and reactive oxygen species act as in situ modulators of follicle maturation and ovulation. Evidence now supports the notion that macrophages are activated by lipid metabolites and further modulation by lipid signaling is a common pathology in atherosclerosis, obesity associated insulin resistance and inflammation arising from metabolic syndrome such as liver steatosis (Prieur et al., 2010), which meat-type AD-hens also manifest (Chen et al., 2006, Pan et al., 2012). However, less is known about the effect of lipid overload on leukocytes in avian species and subsequent impact on ovarian functions.

We previously found that MMP (matrix metalloproteinase) activity and cytokine production were impaired in conjunction with lipotoxic development from peripheral leukocytes in broiler AD-hens (Liu et al., 2014, Pan et al., 2012, Walzem and Chen, 2014). The present study thus sought to determine whether exogenous palmitic acid, a fatty acid shown active in lipotoxic mechanisms (Walzem and Chen, 2014) also induces lipotoxic development in leukocytes of meat-type country chickens. A second aim was to assess subsequent impacts of feed intake on leukocytes and their ability to act in granulosa cell functions related to ovulatory process.

Section snippets

Animal management

Two hundred commercially reared meat-type Red Feather (RF) Taiwan country chickens breeder pullets at age 14 week were purchased from a local breeder farm. All birds were allowed free access to a soy and corn-based breeder pre-lay mash (11.6 MJ/kg metabolizable energy; 15% crude protein). After 2 weeks of adaptation to experimental conditions half of the 16 week old pullets were continued with ad libitum feed intake (AD-hens) with a regular layer mash (11.6 MJ/kg metabolizable energy; 17% crude

Characterization of F1 follicle maturation, inflammation and ovulatory process

In contrast to those of R-hens, F1 follicle granulosa cell progesterone content was significantly reduced in AD-hens coincident with increased NO and proinflammatory IL-1β production (P < 0.05, Fig. 1). AD-hens exhibited significantly reduced gelatinolytic activity in the 55 kDa bands, a collagenase-3-like activity, in both F1 granulosa layer and follicle wall extracts. Similarly, reduced activity was found in F1 follicle wall extracts within the 72 kDa region, the location of a putative gelatinase

Discussion

Ad libitum feeding of meat-type Red Feather country chickens results in dyslipidemia and lipotoxic and inflammatory manifestations that result in reduced egg production similar to those shown in broiler meat-type hens (Chen et al., 2006, Pan et al., 2012, Pan et al., 2014). The present work shows that leukocytes are involved in the ovulatory processes of RF country chickens as was also observed in commercial broiler hens (Liu et al., 2014). While the full nature of the role that leukocytes play

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors appreciated Ms. Yu-En Pan for her technical assistance. This project has been funded in part by the grant of the National Science of Council, Taiwan; NSC100-2321-B-005-008-MY3 and NSC101-2311-B-005-008-MY3, the Council of Agriculture, Taiwan; 96AS-1.2.1-AD-U1, the Ministry of Education, Taiwan, under the ATU plan and Texas AgriLife Research project # 8738.

References (32)

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  • Obesity-associated cardiac pathogenesis in broiler breeder hens: Development of metabolic cardiomyopathy,

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    Few studies have examined premature mortality in adult broiler breeders where concern exists with welfare issues regarding feed restriction to encourage egg production vs. chronic hunger that accompanies this management practice. Our previous works have shown that predictable detrimental metabolic changes (Unger, 2002) accompany lifting of feed restriction that in the short term results in reduced egg production and settable eggs (Chen et al., 2006; Pan et al., 2012; Xie et al., 2012; Liu et al., 2014, 2016; Walzem and Chen 2014). In mammals it is known that obesity and related metabolic disturbances can compromise heart function (Palomer et al., 2013).

1

These authors contributed equally to this work.

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