Elsevier

Theriogenology

Volume 114, 1 July 2018, Pages 116-125
Theriogenology

Improved uterine immune mediators in Holstein cows supplemented with rumen-protected methionine and discovery of neutrophil extracellular traps (NET)

https://doi.org/10.1016/j.theriogenology.2018.03.033Get rights and content

Highlights

  • Discovery of neutrophil extracellular trap formation in bovine endometrial tissue.

  • Supplementing methionine resulted in greater number of glands per uterine tissue at 73 days after calving.

  • Supplementing methionine resulted in higher uterine glandular epithelial cell at 15 days after calving.

  • Supplementing methionine resulted in increased neutrophil infiltration.

Abstract

During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ± 2.0 μM) than cows in MET (30.39 ± 1.6 μM). Cows in CON had greater cystine plasma concentrations (3.62 ± 0.3 μM) than cows in MET (2.8 ± 0.3 μM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity.

Introduction

The increase in nutritional requirements and decreased dry matter intake (DMI) during the transition period results in metabolic and reproductive challenges that can affect the productivity of the cow's future lactation [1]. Implementation of nutritional strategies during the transition from low energy requirements in the dry period to high energy requirements in the lactation period may reduce the metabolic stress of the transition period. A review by Butler [2] described how reproductive diseases and deficiencies, such as retained placenta, metritis, subclinical endometritis, and delayed conception are associated with negative energy balance (NEB) and can lead to decreased fertility. Understanding the changing metabolic requirements of the dairy cow and implementing feeding strategies during the transition period to prevent the nutritional deficiencies that lead to NEB can improve reproductive performance and uterine recovery of the dairy cow after parturition [3].

Methionine is one of the most limiting amino acids for dairy cattle [4]. Rapid degradation of this amino acid in the rumen can result in low absorbance in the small intestine and decreased availability [5]. Previous studies have shown that supplementation of rumen-protected methionine (RPM) increased methionine ruminal bypass, small intestine degradation, and plasma methionine concentrations [5,6]. Additionally, supplementation of RPM leads to increased DMI during the transition period, greater plasma concentrations of sulfur-containing compounds (cystathionine, cystine, homocystine, and taurine), and availability of amino acids (lysine, arginine, tryptophan, threonine, proline, asparagine, alanine, and citrulline) that were once limited as a result of NEB [7,8].

Negative energy balance during the periparturient period is immunosuppressive [9]. According to Sheldon et al. [10], the development of uterine diseases depends on the immunological capacity of the cow, the bacterial species present, and the concentration of bacteria in the bovine uterus. Therefore, uterine disease is expected when NEB and contamination of the uterus are present. In healthy animals, neutrophils are a type of leukocyte that are constitutively present in the reproductive tract. In response to infection, these cells rapidly infiltrate affected tissues and are essential in clearing pathogens as well as dead tissue [11]. Interestingly, an inhibited immune response can result in persistence of the infection and delayed conception as modeled by LeBlanc [11]. Studies have shown that supplementation of RPM can improve the immune status of the cow and increase blood neutrophil function via phagocytosis and oxidative burst activity [12,13]. Increased polymorphonuclear neutrophils (PMN) in the uterus can be an indicator of endometritis [10,14]. However, an increase in neutrophil infiltration during the first two weeks after parturition may be an indicator of improved immune function as hypothesized by LeBlanc [11].

In order to achieve a viable pregnancy and maintain fertility, the uterus endures chemical and morphological changes during the gestation period and after parturition [15]. Development of endometrial glands, termed adenogenesis, consist of the growth of glands that result in secretion of histotroph and other substances that aid in fetal growth and uterine recovery after birth [15]. However, malfunction of the uterus or underdevelopment of the specific glands, may lead to decreased fertility in the future [16]. Nutritional deficiencies can also lead to impaired uterine function, due to persistent infection and reduced immune defenses, which may impact reproductive efficiency [11]. Chronic inflammation, including increased lymphocytic foci, may result in embryonic losses from cows that previously had postpartum uterine diseases [17]. Histotroph secretions are mainly composed of serine and methionine, therefore, supplementation of methionine may increase amino acid availability for histotroph secretion and lead to better fetal development [18].

Presence of antioxidant factors, such as Glutathione Peroxidase 1 (GPX) and Superoxide Dismutase 1 (SOD), may decrease damaging reactive oxygen species and impact uterine physiology [11,19]. Results from a previous study reported that GPX and SOD are commonly expressed in the canine endometrium during the estrus cycle and in different epithelial structures, suggesting that these factors play an important role in the cycling uterus [19]. Likewise, gene expression of GPX and SOD are commonly found in the bovine oviduct, with an up-regulation of GPX genes occurring before ovulation, suggesting that these antioxidants may play a role in successful fertilization and implantation [20].

Therefore, the objectives of this study were to determine the effect of feeding RPM on neutrophil infiltration, uterine morphology, and expression of GPX and SOD, as well as to confirm neutrophil extracellular trap (NET) formation in the endometrium of Holstein cows during the postpartum period.

Section snippets

Materials and methods

The following procedures were approved by the University of Illinois (Urbana-Champaign) Institutional Animal Care and Use Committee (IACUC).

Results

Blood plasma amino acid and metabolite concentrations are in Table 1. Plasma from cows in MET (30.39 ± 1.6 μM) had greater (P < 0.01) methionine concentrations than cows in CON (18.05 ± 2.0 μM). No treatment differences (P > 0.10) were observed for any other dispensable or indispensable amino acids in plasma. A tendency for a treatment by time interaction was detected for arginine (P = 0.11) and glutamine (P = 0.09; Fig. 1).

Plasma cystine concentrations were greater (P < 0.01) for cows in CON

Discussion

The aim of this study was to evaluate the impact of feeding RPM on plasma amino acid and metabolite concentrations, endometrial glandular morphology, uterine neutrophil infiltration, neutrophil function (NET formation), and expression of SOD, GPX, and NE in the endometrial lining of dairy cows during the postpartum period. We postulated that supplementation of RPM, a limiting amino acid in dairy cattle, would increase the availability of other essential amino acids and metabolites leading to

Acknowledgements

This work was partially supported by the USDA National Institute of Food and Agriculture (Washington, DC; W-3132) and Adisseo (Commentry, France). The authors also extend thanks to all the University of Illinois researchers, veterinarians, and the Dairy Research Unit staff involved in collection of the data.

References (53)

  • J.S. Osorio et al.

    Supplemental Smartamine M or MetaSmart during the transition period benefits postpartal cow performance and blood neutrophil function

    J Dairy Sci

    (2013)
  • M.C. Lucy et al.

    Lymphocytic foci in the endometrium of pregnant dairy cows: characterization and association with reduced placental weight and embryonic loss

    Theriogenology

    (2016)
  • C. Santos et al.

    Distribution of superoxide dismutase 1 and glutathione peroxidase 1 in the cyclic canine endometrium

    Theriogenology

    (2016)
  • D.A.V. Acosta et al.

    Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows

    Theriogenology

    (2016)
  • E.C. Titgemeyer et al.

    Estimation of lysine and methionine requirements of growing steers fed corn silage-based or corn-based diets

    J Dairy Sci

    (1988)
  • S.P. Stabler et al.

    α-Lipoic acid induces elevated S-adenosylhomocysteine and depletes S-adenosylmethionine

    Free Radic Biol Med

    (2009)
  • M. Balaghi et al.

    Pancreatic one-carbon metabolism in early folate deficiency in rats

    Am J Clin Nutr

    (1993)
  • J. Cheng et al.

    Effects of rumen-protected γ-aminobutyric acid on performance and nutrient digestibility in heat-stressed dairy cows

    J Dairy Sci

    (2014)
  • K.A. Piez et al.

    The conversion of lysine to hydroxylysine and its relation to the biosynthesis of collagen in several tissues of the rat

    Biol Chem

    (1957)
  • J.M. Huzzey et al.

    Prepartum behavior and dry matter intake identify dairy cows at risk for metritis

    J Dairy Sci

    (2007)
  • A. Chapwanya et al.

    Endometrial biopsy: a valuable clinical and research tool in bovine reproduction

    Theriogenology

    (2010)
  • S. Ohtani et al.

    Histological changes in bovine endometrium during the estrous cycle

    Theriogenology

    (1993)
  • A. Chapwanya et al.

    Histopathological and molecular evaluation of Holstein-Friesian cows postpartum: toward an improved understanding of uterine innate immunity

    Theriogenology

    (2009)
  • O.I. Azawi

    Postpartum uterine infection in cattle

    Anim Reprod Sci

    (2008)
  • G.S. Dhaliwal et al.

    Some aspects of immunology of the bovine uterus related to treatments for endometritis

    Anim Reprod Sci

    (2001)
  • R. Kasimanickam et al.

    Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows

    Theriogenology

    (2004)
  • Cited by (20)

    • Methionine supply during the peripartum period and early lactation alter immunometabolic gene expression in cytological smear and endometrial tissue of holstein cows

      2021, Theriogenology
      Citation Excerpt :

      Regarding the uterine health parameters, cows that were fed RPM had greater percentage of polymorphonuclear (PMN) cells at 15 DIM, but lower at 30 DIM and at 73 DIM, when compared with cows that were not fed RPM [21]. Moreover, cows that were fed RPM tended to be classified as not having subclinical endometritis at 30 DIM, in comparison with cows that were not fed RPM [21]. Results for mRNA expression in cytological smear samples are in Fig. 1 and Table 2.

    • Symposium review: Nutrition strategies for improved health, production, and fertility during the transition period

      2020, Journal of Dairy Science
      Citation Excerpt :

      Higher proportions of PMN from endometrial cytology of dairy cows receiving RPM at 15 DIM but lower PMN at 72 DIM than cows not receiving RPM is an indication of improved uterine environment (Stella et al., 2018). Results from Stella et al. (2018) agree with LeBlanc (2014), who proposed that healthy cows (i.e., without endometritis) can build an immunological response early in lactation. Perhaps the most detrimental effect of NEB on reproductive performance is delayed return to cyclicity.

    View all citing articles on Scopus
    View full text