Metabolic biomarkers, body condition, uterine inflammation and response to superovulation in lactating Holstein cows

Journal Pre-proof Metabolic biomarkers, body condition, uterine inflammation and response to superovulation in lactating Holstein cows R. Kasimanickam, V. Kasimanickam, J.P. Kastelic, K. Ramsey PII:

S0093-691X(20)30102-3

DOI:

https://doi.org/10.1016/j.theriogenology.2020.02.006

Reference:

THE 15386

To appear in:

Theriogenology

Received Date: 4 February 2019 Revised Date:

10 November 2019

Accepted Date: 4 February 2020

Please cite this article as: Kasimanickam R, Kasimanickam V, Kastelic JP, Ramsey K, Metabolic biomarkers, body condition, uterine inflammation and response to superovulation in lactating Holstein cows, Theriogenology (2020), doi: https://doi.org/10.1016/j.theriogenology.2020.02.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

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Metabolic biomarkers, body condition, uterine inflammation and response to superovulation in

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lactating Holstein cows

3 Kasimanickam R*1, Kasimanickam V12, Kastelic JP3, Ramsey K1

4 1

5 6 7 8

Department of Veterinary Clinical Sciences, 2Center for Reproductive Biology,

College of Veterinary Medicine, Washington State University, Pullman, WA 99164 3

Department of Production Animal Health, University of Calgary, Faculty of Veterinary Medicine, Calgary, AB, Canada

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Corresponding author: [email protected]

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Abstract

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The objective was to determine associations between response to superovulation and

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body condition, subclinical endometritis and circulating metabolic biomarkers [adiponectin,

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leptin, insulin, IGF1, tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL6, and urea] in

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lactating dairy cows. Ten multiparous lactating Holstein cows in each body condition score (1 to

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5; 1 emaciated; 5 obese) category (BCSC) 2.00 to < 2.50 (BCSC1), 2.50 to < 3.00 (BCSC2),

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3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00 (BCSC5) groups (total n=50)

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were randomly selected and superovulated, timed artificially inseminated with frozen-thawed

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semen from three sires and embryos collected (n = 50 collections). At embryo collection, blood

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samples and embryo recovery fluid were collected for determination of metabolic markers and

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presence of subclinical endometritis (lavage technique; > 6% PMN). In total, 379 embryos were

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collected (average of 7.6 embryos per superovulation). Mean numbers of total ova and embryos

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was greater for cows in BCSC2, BCSC3 and BCSC4 groups compared with cows in BCSC1 and

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BCSC5 groups (P<0.01). Total number of transferrable embryos were greater for cows in BCSC

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2 and BCSC3 groups compared with cows in BCSC1, BCSC4 and BCSC5 groups (P<0.01).

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Mean number of total ova and embryos and of transferrable embryos was higher for cows with 0

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or 1 to 6% PMN compared to cows with >6% PMN (P<0.01). In addition, there was a quadratic

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association between blood urea nitrogen concentrations and % transferrable embryos (r2=0.85;

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P<0.05) and between BCS and % transferrable embryos (r2=0.73; P<0.05). Circulating

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adiponectin, leptin, insulin, IGF1 and TNFα were greater in cows with moderate to good body

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condition compared to thin or obese cows (P<0.05). Circulating adiponectin, leptin, IGF1 and

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insulin were greater in normal cows (≤ 6% PMNs), whereas, TNFα and IL1β and IL6 were

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greater in cows with subclinical endometritis (P<0.05). In conclusion, BCS and subclinical

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endometrial inflammation were associated with superovulatory response and embryo quality.

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Further, circulating metabolic biomarkers were associated with superovulatory response and

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embryo quality, likely due to donor’s metabolic status and uterine environment. Optimizing

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superovulatory responses and embryo quality in lactating dairy cows requires management of

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nutrition and uterine health.

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Keywords: Dairy cows; Superovulation, Metabolic biomarkers; Body condition; subclinical

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endometritis;

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1 Introduction

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Early embryo development during the first week after fertilization in modern lactating

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dairy cows is suboptimal, with only ~50% of embryos reported viable by Day 7 post fertilization

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[1]. Factors affecting dairy cow fertility include genetics, diseases and management, likely by

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influencing uterine environment, oocyte quality and embryo development. Diet, milk production,

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energy status and blood urea concentrations (BUN) have been associated with reduced fertility in

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dairy cows [2-6]. Elevated BUN resulting from excess rumen degradable protein or dietary urea

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decreases uterine luminal pH in cows and ewes, increasing embryonic loss and reducing

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pregnancy rate [5-9]. Peripartum loss in body condition alters metabolic profiles and increases

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incidence of metabolic diseases in postpartum dairy cows; these aberrant metabolic profiles may

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have negative effects on the uterus and developing embryo later in lactation, when rebreeding

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commences [10].

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Adipokines [adiponectin, leptin, tumor necrosis factor (TNF) α and interleukins (IL)],

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secreted by adipose tissue, communicate with brain and peripheral tissues [11,12]. Adiponectin

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regulates carbohydrate and lipid metabolism and has both anti-inflammatory and anti-obesity

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properties, whereas TNFα and IL6 are pro-inflammatory cytokines [13]. Leptin regulates feed

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intake as well as metabolic and endocrine functions and also modulates immunity, inflammation

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and hematopoiesis [14]. Adiponectin and leptin together normalize insulin action in severely

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insulin-resistant animals [15]. Leptin also improves insulin resistance and reduces

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hyperlipidemia. Adiponectin production is stimulated by agonists of peroxisome proliferator-

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activated receptor-gamma (PPARG), an action that may contribute to the insulin-sensitizing

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effects of this class of compounds [16]. Production of adiponectin, leptin and insulin in cattle are

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influenced by nutritional status. Assessing body condition of cows is a practical method of

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evaluating nutritional status of the cattle.

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Adiponectin and its receptor 1 and 2, leptin, IGF and insulin have roles in early embryo

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development. Adiponectin has direct effects on ovarian function and early embryo development

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[17] and also has indirect effects on embryos via modulation of insulin and IGF-I [18]. In

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oocytes retrieved from calf ovaries, adding leptin to the in vitro maturation medium enhanced

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meiotic maturation, embryo development and embryo quality [19]. In developing bovine

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blastocysts, insulin blocked apoptosis and acted as a survival factor, whereas IGF-I stimulated

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blastocyst development and increased blastomere numbers. Collectively, these studies

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highlighted associations between these metabolic biomarkers and embryo quality.

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Our objective was to determine associations between circulating metabolic biomarkers

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[adiponectin, leptin, insulin, IGF1, tumor necrosis factor (TNF) α, interleukins (IL) 1β and 6, and

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urea], body condition, uterine inflammation and superovulation response in lactating dairy cows.

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2 Materials and methods

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This study was performed in accordance with the ethics, standard operating procedure, handling

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and use of animals, collection and use of biomaterials for research.

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2.1 Cows

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Ten multiparous lactating Holstein cows (second to fifth lactation) in each body condition

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score (1 to 5; 1 emaciated; 5, obese) category (BCSC) 2.00 to < 2.50 (BCSC1), 2.50 to < 3.00

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(BCSC2), 3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00 (BCSC5) (total n=50

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cows) were randomly selected as embryo donors. Each cow was superovulated once and

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embryos collected. At enrolment, cows were between 60 and 90 days in milk and were housed in

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free-stall barns. Cows were not submitted for breeding prior to enrolment. Cows were fed twice

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daily a total mixed ration to meet or exceed dietary requirements for lactating Holstein cows

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weighing 550 to 800 kg and producing 25 to 35 kg of 3.5% fat-corrected milk.

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2.2 Superovulation

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A schematic presentation of superovulation and embryo collection is shown (Figure 1).

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Briefly, on Day 0, all donor cows were given a progesterone-releasing intravaginal insert (CIDR;

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1.38 g of progesterone; Eazi-Breed™ CIDR® Cattle Insert; Zoetis Animal Health, New York,

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NY, USA) and gonadorelin hydrochloride (GnRH; 2 mL (100 µg), im, Factrel®; Zoetis Animal

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103

Health). On Day 4 (84 h after GnRH injection), superovulation with Folltropin-V® (Follicle

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stimulating hormone (FSH) equivalent to 400 mg NIH-FSH-P1; im; Bioniche Animal Health,

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Athens, GA, USA) was initiated, with twice-daily administration of a decreasing dose over 4.5 d.

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Dinoprost (PGF2α; 5 mL (25 mg) im; Lutalyse® sterile solution; Zoetis Animal Health) were

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concurrently administered with the last two FSH injections and progesterone inserts removed

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concurrent with the penultimate FSH treatment (Day 7 PM). Ovulation was induced with GnRH

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(100 µg im; Zoetis Animal Health) 44 h after progesterone insert removal and cows were fixed-

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time artificially inseminated 12 and 24 h after GnRH injection. The sires (n=3) were randomly

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assigned to donor cows. The sire conception rate (SCR) score of all three sires that were used to

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inseminate cows were + 4.

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2.3 Assessment of number of corpora lutea

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One clinician assessed number of corpora lutea (CL) by transrectal ultrasonography

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(Aloka 500, Sysmed Lab, Inc., Chicago, IL, USA) with 5 MHz, linear-array transducer on Day 7,

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immediately before performing the embryo flushing procedure.

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2.4 Embryo collection

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Embryos were collected non-surgically from donor cows on Day 7 after second AI. A

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sterile 16 Fr. two-way Foley catheter with a 5 cm3 balloon (Agtech Inc., Manhattan, KS, USA)

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was positioned in the uterine horn and embryo collection medium (Agtech Inc.) was introduced

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by gravity flow through “Y”-junction tubing. Each uterine horn was flushed individually with

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~200 to 250 mL of medium (3 to 5 flushes per horn) which was passed through an Em-Con

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Embryo Collection Filter (Agtech Inc.) and a small volume of recovered fluid retained in the

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filter. Recovered fluid was transferred from embryo filter into flat, gridded search petri dishes

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and examined under a stereoscope for presence of embryo. Once identified, embryos were

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transferred to another petri dish with holding medium and evaluated for quality and stage of

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development, as described [20]). For consistency, cows with zero response to superovulation

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were also flushed in the same manner for determination of %PMN. Zero response was defined as

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cows with no CL and zero embryo yield following superovulation treatment.

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2.5 Endometrial samples

136 137

Evaluation of endometrial cytology was done as described [21], with some modifications.

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Briefly, 10 mL of clear embryo recovery fluid following uterine flushing was transferred to a 10

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mL polystyrene centrifuge tube (Corning, Corning, NY, USA) without preservatives. Samples

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were centrifuged at 800 × g for 10 min and supernatant discarded. A drop of sediment was

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streaked on to a clean microscopic slide and air-dried. Slides were fixed with cytofixative

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(Cytoprep; Fishers Scientific) and stained with modified Giemsa stain (Protocol-Hema3;

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Biochemical Sciences, Swedesboro, New Jersey, USA). A total of 300 cells were counted under

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a microscope (x 400 magnification) to determine % PMN. To determine the correlation between

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the number of PMN and superovulation response, we categorized %PMN in three groups %

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PMN – 0, % PMN - 1 to 6 and %PMN - >6.

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2.6 Blood metabolites

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2.6.1 Blood sampling and BUN analysis

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On day of embryo collection, blood samples were collected by coccygeal venipuncture

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into evacuated tubes (Becton Dickinson, Franklin Lakes, NJ, USA), held at ambient temperature

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for 5 min then placed on ice, centrifuged at 1200 g (5 ºC) for 15 min within 2 h after collection

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and serum separated and frozen (-20 °C). Samples were subsequently thawed, and BUN

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concentrations measured with ELISA (MBS9376646; MyBioSource, San Diego, CA USA), in

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accordance with manufacturer’s instructions.

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2.6.2 Biomarkers analyses

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Serum cytokines concentrations were determined as described [22]. All primary and

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secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz

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Biotechnology, CA, USA), unless otherwise stated. Briefly, 96-well plates were pre-coated with

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standards and samples for all serum cytokines and kept at 4 °C for at least 24 h. For biomarker

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determination, 100 µL of respective primary antibodies (Supplementary File, Table 1),

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recommended for use in cattle were added to the respective 96-well plates. Corresponding

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peptides or proteins (Supplementary File, Table 1) used for antibody production were used to

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prepare standards. Ranges for standard dilution and serum dilution for each protein target were

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determined from a pre-run of standards and at least three samples. In short, at least eight

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dilutions of each sample (1 in 10, 1 in 50, 1 in 100, 1 in 200, 1 in 400, 1 in 600, 1 in 800 and 1 in

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1000) were used to make dilution curve and compared to the regression line of the standard [22-

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25]. Then, the serum dilution paralleling the regression line of standard was selected. Standard

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dilution ranges were carefully chosen to include lowest and highest readings of all samples [22-

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25]. The shape of the regression line and serum dilution used was different for each target. Each

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96-well plate had one set of standards and samples in triplicate. The ELISA plates were coated

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with 100 µL of standards or samples. Non-specific protein binding was blocked by adding 150

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µL of either 2% donkey or goat serum in PBS to each well (depending on the species used to

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raise secondary antibodies). The plate was left on a rocking platform at room temperature for 60

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min with blocking buffer. Wash buffer was prepared with 0.05% tween20 in PBS. Following

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non-specific protein blocking, samples were incubated (60 min) with diluted primary antibodies.

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Antibody concentrations were initially 200 µg/mL, with dilution to 1:100 before incubation.

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After washing with wash buffer, 100 µL of secondary antibodies conjugated with horse radish

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peroxidase (Supplementary File, Table 1) was added to each well and incubated for 60 min.

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After washing with buffer, 100 µL of reagent containing the substrate of acetyl cholinesterase

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(N301, Thermo Scientific, Logan, UT, USA) was added for the enzymatic reaction until color

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development and then 50 µL of stop solution (N600, Thermo Scientific) was added. Plates were

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read at 450 nm using a Glomax®-Multi Detection System (Promega, Madison, WI, USA) and

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serum concentrations of adiponectin, leptin, IGF1, insulin, TNFα, IL6, IL1β were calculated

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from standard curves (using mean readings and dilution factors). Intra- and inter-assay CVs were

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6.9 and 9.8% for adiponectin, 7.1 and 10.4% for leptin, 7.1 and 10.8% for IGF1, 9.0 and 10.2%

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for insulin, 7.8 and 12.2% for IL6, 9.6 and 12.3% for IL1β, and 8.5 and 13.2% for TNFα,

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respectively.

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2.7 Statistical analyses

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Data were analyzed with Statistical Analysis System (SAS) version 9.4 (SAS Institute,

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Inc., Cary, NC, USA) software and presented as mean ± SEM. Datasets were tested for normality

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distribution by Komogorov-Smirnov test and were log 10 or arcsine transformed as needed. Cow

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was used as the experimental unit for embryo number and quality. ANOVA and multiple

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comparisons by Tukey test were used for the following: 1) effect of body condition on number of

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corpora lutea, total ova and embryos and transferable embryos and embryo stages; 2) association

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of body condition (thin, BCSC1; moderate to good, BCSC2, BCSC3 and BCSC4, obese,

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BCSC5) on adipokines; 3) effect of uterine inflammation category at embryo recovery on

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number of corpora lutea and number of embryos classified to various quality grades; 4)

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association of uterine inflammation on adipokines; 5) effect of sire on uterine inflammation; 6)

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effect of sire on total ova and embryos and transferable embryos; and 6) effect of BUN on

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transferrable embryos (%, embryo was used as experimental unit). Correlations between body

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condition and BUN concentrations and between BUN concentrations and transferrable embryo

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were assessed by Pearson Correlation Coefficient cows with 0 and 1 to 6% PMN were combined

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and defined as having no subclinical endometritis. For all analyses, P > 0.05 was considered non-

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significant.

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3 Results

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The %PMN in endometrial samples ranged from 0 to 54%. At embryo collection on Day

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7, 26 (52.0%), 13 (26%) and 11 (22%) cows had 0, 1 to 6 and >6 %PMNs, respectively. Sire did

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not influence %PMN (P>0.1) A total of 379 ova and embryos were collected from 50 embryo

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collections with 7.6 (±1.6) ova and embryos per superovulation. Percentage of transferrable

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embryos (Grade 1, 2 and 3) was 76.8 (291/379) and percentage of degenerate

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embryos/unfertilized oocytes was 23.2 (88/379). Frozen thawed semen from Sires 1, 2 and 3

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were used to inseminate 17, 17 and 16 donors, respectively. Number and percentage of total ova

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and embryos were not different among sires (Sire 1, 31.4% (119/379), Sire 2, 32.7% (124/379)

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and Sire 3, 35.9% (136/379); P>0.1). Further, transferable embryos were not different among

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sires (Sire 1, 31.3% (91/291), 34.0% 99/291, 34.7% (101/291); P>0.1).

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3.1. Effect of cows’ body condition on embryo number and quality

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Considering body condition score at the beginning of superovulation procedure, mean

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number of total ova and embryos was greater for cows in BCSC2, BCSC3 and BCSC4 groups

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compared with cows in BCSC1 and BCSC5 groups (P<0.01; Table 1). Total number of

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transferrable embryos were greater for cows in BCSC2 and BCSC3 groups 2 compared with

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cows in BCSC1, BCSC 4 and BCSC5 groups (P<0.01). Number of transferrable embryos was

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not different between cows in BCSC1, BCSC 4 and BCSC5 groups (P>0.1). Mean number of

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morulae collected was greater for cows in BCSC3 group compared with cows in BCSC1,

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BCSC2, BCSC4 and BCSC5 groups, whereas number of blastocysts recovered were greater for

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cows in BCSC2, BCSC3, BCSC4 groups compared to cows in BCSC1 and BCSC5 groups.

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Percentages of superovulation that yielded no ova and embryos for cows in BCSC1, BCSC2,

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BCSC3, BCSC4 and BCSC5 groups were 20, 10, 10, 20 and 40%, respectively. There was a

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quadratic relationship between BCS and % transferrable embryo (r2=0.73; P<0.05).

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3.2. Effect of endometrial PMN percentage Considering PMN categories at embryo flush, mean number of total ova and embryos

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was higher for cows with 0 or 1 to 6% PMN compared to cows with >6% PMN (P<0.01; Table

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2). Mean number of transferrable embryos were greater for cows with 0% and 1 to 6% PMN

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compared to cows with >6% PMN (P<0.01; Table 2). Mean number of morula and blastocysts

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were recovered from cows with < 6% PMN were greater compared to cows with > 6% PMN.

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Percentages of superovulation that yielded no embryos for cows with 0, 1 to 6 and >6% PMN

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were 20, 20 and 60%, respectively.

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3.3. Effect of BUN on embryo quality

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There was a linear association between body condition score and BUN concentration

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(r2=0.82; P<0.05; Figure 2) and a quadratic relationship between BUN concentrations and %

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transferrable embryos (r2=0.86; P<0.05).

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3.4 Association of body condition, uterine inflammation and adipokines, and effect of adipokines

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on embryo quality

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Serum concentrations of adipokines (except ILs), IGF1 and insulin were greater in cows

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with moderate to good body condition (P<0.05; Figure 3). Serum leptin was lower in thin

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compared with moderate to obese cows (P<0.05). Serum adiponectin, leptin, IGF1 and insulin

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were greater in cows with < 6% PMNs (P<0.05), whereas, TNFα and ILs were greater in cows

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with > 6 %PMN (P<0.05).

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Considering body condition of donor cows, there were quadratic associations between

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metabolic biomarkers and transferable embryos, and ‘r’ varied from 0.82 to 0.94 (P<0.05).

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Considering subclinical endometritis of donor cows, there were positive correlations between

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transferable embryos and adiponectin, leptin, insulin and IGF1, and ‘r’ varied from 0.52 to 0.85

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(P<0.05). Furthermore, there was negative correlation between transferable embryos and TNFα,

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IL1β and IL6 (P<0.05).

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4 Discussion

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Our objective was to determine associations among circulating metabolic biomarkers

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[adiponectin, leptin, insulin, IGF1, tumor necrosis factor (TNF) α, IL1β, IL6, and urea], body

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condition, subclinical endometritis and embryo quality in lactating dairy cows. Results observed

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in the current study was supported by previous reports regarding associations among BUN, BCS

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and embryo quality [6,7]. Furthermore, our data increased understanding of how embryo quality

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is influenced by adipokines, BCS and uterine inflammation in modern dairy cows [3,26,27].

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Reproductive efficiency in lactating dairy cows is a long-standing concern. Nutritional

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demands and inherent stress during early lactation are important contributors to failure to re-

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establish cyclicity, ovulate, and/or become pregnant [28-31]. Energy status has profound effects

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on metabolic homeostasis and reproductive performance of dairy cows [28-31]. Negative energy

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balance and accompanying loss of body condition is a major cause of ovarian inactivity during

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the first several weeks postpartum; thin cows had a greater incidence of anovulation than those in

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moderate to good body condition [32]. An adequate rise in post- ovulatory progesterone

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concentrations drives ‘normal’ temporal changes in the endometrium. This prepares the

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endometrium to release histotroph, a source of nutrition for embryos prior to attachment. A

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positive change in BCS between calving and breeding increased proportion of cows with >1

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ng/mL progesterone after AI [32-35]. These studies highlighted effects of BCS on progesterone

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concentrations, which are vital for uterine receptivity and fertility.

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Blood urea nitrogen concentration is a well-established measure for protein digestion and

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catabolism; high-protein diets and elevated BUN have been implicated in altered uterine pH,

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resulting in decreased reproductive performance in dairy cows [8,16,36,37]. Decreased fertility is

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likely a consequence of toxic effects of urea on oocytes and/or embryos [36,37]. Although

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number, quality and stage of development of recovered embryos were similar for cows with

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moderate or high PUN [38], transfer of frozen-thawed embryos to virgin heifers from moderate

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PUN donor cows increased pregnancy rate (35%; P<0.02) compared to transfer of embryos from

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high PUN donor cows (11%) [38]. Interestingly, pregnancy rate was not affected by neither

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recipient diet nor interaction of donor and recipient diets (P>0.05). Therefore, high PUN

300

concentrations in lactating dairy cows decreased embryo viability through effects exerted on the

301

oocyte or embryo before recovery (7 d after insemination). In the current study, there were

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quadratic associations between body condition score and BUN; BCS and % transferable

303

embryos; and between BUN and % transferrable embryos.

304

Effects of BCS, diets and biological markers on embryo yield and quality have been

305

reported. For example, BCS was closely associated with IGF and bST and embryo production in

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superovulated cows [39]. Giving donors bST increased embryo yield, attributed to increased

307

IGF1 concentrations in follicular fluid and peripheral plasma [40,41]. In a 2 x 2 factorial study of

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factors affecting embryo quality, there was an interaction between feed (high-energy diets or

309

feed restriction) and low or high LH during superovulation; therefore, it was concluded that both

14

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metabolic and endocrine status affect superovulatory success. Overfeeding results in high

311

peripheral concentrations of both LH and insulin, with negative effects on fertilization and

312

embryo quality [42]. Similarly, there was an inverse relationship between overfeeding and in

313

vivo embryo production [43,44]. In a study of the association of BCS with % transferable

314

embryos from fertilized ova and % transferable embryos from total ova [45], elevated but not

315

lowered BCS was associated with decreased superovulatory response, as measured by number of

316

corpora lutea (P<0.0001), total ova and embryos recovered (P=0.0004), and number of fertilized

317

ova (P< 0.0001). However, no differences in number of transferable embryos were detected,

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primarily because percentage of fertilized ova that resulted in transferable embryos was greater

319

(P<0.0001) in donors with higher BCS. Total number of ova and embryos were greater, but

320

number of transferable embryos lower in their study compared to the current study.

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Subclinical endometritis reduces fertility by creating an unreceptive uterine environment

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for developing embryos, possibly causing early embryonic loss [46,47]. In vitro production of

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embryos in fluid consistent with an inflamed uterine environment resulted in poor early

324

embryonic development and increased embryonic loss [48], consistent with a greater proportion

325

of PMN in the uterine lumen negatively affecting number of transferable embryos in the present

326

study. Several genes expressed in the endometrium of cows with subclinical endometritis during

327

early embryonic development plausibly affect downstream pathways essential for early

328

embryonic development in a negative way, resulting in poor embryo quality [48].

329

In the current study, adipokines (except ILs), IGF1 and insulin were greater in cows with

330

moderate to good body condition. Circulating adiponectin, leptin, IGF1 and insulin were greater

331

in cows with < 6% PMNs, whereas, TNFα and ILs were greater in cows with > 6%PMN.

332

Interestingly, more transferable embryos were produced by cows with moderate to good body

15

333

condition and in cows without subclinical endometritis. We inferred metabolic biomarkers had

334

direct or indirect effects on superovulatory responses and embryo quality.

335

Adiponectin and its receptors AdipoR1 and AdipoR2 are present in bovine embryos

336

[49,50]. Furthermore, inclusion of adiponectin during oocyte maturation or embryo culture

337

promoted blastocyst formation in swine embryos [51]. However, in a bovine study, addition of

338

adiponectin during in vitro maturation did not affect oocyte maturation, embryo cleavage, or

339

development to the blastocyst stage [49]. Furthermore, adiponectin knock-out mice are fertile

340

[52]. Collectively, these studies confirmed species to species variation and that adiponectin was

341

not essential for fertility and/or embryo development. However, adiponectin can have an

342

important complementary role in regulation of female reproductive function. Adiponectin has

343

been implicated in modulation of ovarian and endometrial function, influencing periovulatory

344

remodeling of the ovarian follicle, steroid synthesis/secretion as well as energy supply and

345

inflammatory responses of endometrial cells [22,49,50,53-56].

346

In the current study, circulating leptin was lower in thin cows versus those that were in

347

moderate to obese condition (P<0.05). Leptin is involved in regulation of ovarian function,

348

oocyte maturation and pre‐implantation embryo development [57-59]. Leptin receptors have

349

been detected in porcine [60] and bovine embryos [61] and addition of leptin to in vitro

350

maturation medium enhanced meiotic maturation, and embryo development from calf oocytes,

351

improving embryo quality [19]. Furthermore, leptin administration during superovulation

352

increased embryo quality [62]. Goats given leptin had higher yield of transferable embryos

353

(P<0.005) with fewer apoptotic cells in blastocysts and fewer degenerated embryos (P<0.001)

354

compared to a control group [62]. Embryonic development also depends on cytoplasmic

355

maturation that involves organelle reorganization and storage of mRNAs, proteins and

16

356

transcription factors. Calf oocytes can resume meiosis and progress to MII after in vitro

357

maturation [63], but cytoplasmic maturation in vitro is generally compromised, reducing

358

developmental competence compared to oocytes from adults. Leptin may enhance cytoplasmic

359

maturation of oocytes and promote embryo development into blastocysts [19].

360

In cattle, IGF1 has an important role in follicular growth, oocyte competence acquisition

361

and embryo viability [41,64]; however, IGF1 can have either positive or negative effects on

362

embryo yield or viability, depending on its concentration during superovulation. Plasma IGF1

363

concentrations before superstimulation did not predict number and quality of embryos; however,

364

there was an association between IGF1 concentration at estrus and on day of embryo collection

365

with total grade 1 embryo yield [65]. In general, physiological concentrations appeared to

366

improve embryo quality, mainly due to mitogenic and anti-apoptotic activities of IGF1 [66].

367

However, high IGF1 concentrations are associated with decreased embryo viability and a

368

concomitant increase of apoptosis. In the current study, IGF1 concentrations had a quadratic

369

association with transferable embryos, indicating that extreme IGF1concentrations were

370

detrimental to embryo number and quality.

371

Insulin has crucial functions in energy metabolism; low circulating insulin concentrations

372

in dairy cows indicate energy imbalance, especially during postpartum period. Negative

373

consequences of low circulating insulin concentrations include delayed resumption of

374

postpartum cyclicity and unfavorable conditions for early embryonic development. Insulin has

375

been used for many years as a stimulatory factor for in vitro cell culture and for in vitro embryo

376

production. However, there are variable effects of insulin on early embryonic development, with

377

some studies reporting changes in blastocyst cell numbers [67], whereas other studies did not

378

detect any effects on blastocyst rate [67-71], with one report of developmental stimulation until

17

379

the morula stage [71]. Responses to exogenous insulin vary among species. There are direct

380

effects of insulin on expression of anti Müllerian hormone (AMH) in women [72], whereas

381

insulin supplementation in single-step embryo culture medium from day 0 through days 5 or 6

382

improved development of human embryos [73]. In vitro development of murine embryos may be

383

enhanced with exogenous insulin, although it had no detectable beneficial effect on bovine

384

embryo development [74]. Insulin treatment decreased apoptosis, implying it may function as a

385

mitogen or anti-apoptotic factor during early bovine embryonic development [75]. Further,

386

reduced fertility in obese dairy cows may be connected to impaired insulin sensitivity [76]. In

387

general, either high or low insulin concentrations can be deleterious to fertility and early

388

embryonic development. In the current study, thin or obese cows yielded lower % transferable

389

embryos, associated with low and high insulin concentrations respectively, compared to

390

moderate to good conditioned cows.

391

Pro-inflammatory cytokines IL1β, IL6 and TNFα were increased both in circulation [22]

392

and in endometrium of cows with uterine inflammation [48]. In a previous study, circulating

393

concentrations of TNFα, IL1β and IL6 were greater in postpartum cows with uterine

394

inflammatory conditions that also had lower BCS [22]. In the present study, TNFα, IL1β and IL6

395

were elevated in cows with subclinical endometritis at embryo collection. It should be noted that

396

%PMN cut-off in the previous study was 18% [48] whereas, in the current study it was 6%. In

397

addition, there was a quadratic association between TNFα, IL1β, IL6 and BCS. It should be

398

noted that 18% PMN cut-off for sampling at 21 to 34 DIM and 10% PMN cut-off for sampling at

399

35 to 49 DIM were used [77]. It has been reported that as DIM increases %PMN cut-off

400

decreases [77]; as sampling in this study was done >60 DIM, the cut-off value was 6% PMN.

18

401

Elevated serum pro-inflammatory cytokines including TNF and IL1β are associated with

402

fewer viable inner cell mass cells in blastocysts [78,79,81,82]. In contrast, proinflammatory

403

cytokines secreted by an embryo can regulate release of embryo-trophic factor from the uterus

404

[80]. In addition, early embryo derived factors suppress expression of TNFα and IL1β in uterus.

405

During early pregnancy, there is evidence for production of IL1β by both embryo and

406

reproductive tract. IL1β promotes embryonic development through increased secretion of

407

embryonic leukemia inhibitory factor (LIF) [81]. Maximum stimulatory effects of IL1β on

408

growth of embryos was greatest at a concentration of 0.1 ng/ml [82]. In addition, IL1β may

409

inhibit some metabolic pathway or gene transcription; perhaps limited inhibition is beneficial for

410

development, whereas more profound inhibition reduces development.

411

Previous studies [42,45] reported a greater number of flushes resulted in zero ova and

412

embryos following superovulation compared to the current study. This apparent difference

413

between studies was plausibly due to selection pressure towards production traits in our cow

414

population. When donor cows were highly preselected for production, 22.6% of all flushes

415

resulted in zero ova and embryos [83].

416

From a genetic perspective, number of total ova and embryos describes a cow’s

417

performance and only depend on her genetic effect, whereas for traits describing embryonic

418

survival, paternal effect is also relevant [84]. High SCR bulls produced a lower percentage of

419

unfertilized oocytes and fewer degenerated embryos compared to low SCR bulls [85]. In the

420

current study, there was no difference among sires for total number of ova and embryos, or

421

percentage of transferable embryos. However, only high SCR (+4) sires were used.

422 423

4.1 Conclusions

19

424 425

In conclusion, embryo production and development were influenced by body condition

426

score, subclinical endometrial inflammation and various metabolic biomarkers. Further,

427

circulating adipokines, IGF1, insulin and urea may affect embryo quality, due to their roles in

428

donor’s metabolic status and uterine environment. Nutrition and uterine health of lactating dairy

429

cows should be well managed to promote superovulatory response and embryo quality.

430 431

Conflict of interest

432 433

The authors declare no conflict of interest.

434 435

Authors’ contributions

436 437

RK and VK contributed to conception and design of the study; RK collected samples and

438

data; RK, VK and KR performed analysis, interpretation, drafting of the article. RK, VK and JK

439

revising it critically for important intellectual content, and RK, VK, KR, JK approved the final

440

version.

20

441

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1412.

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658

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30

659

Table 1. Mean (± SEM) reproductive data in superovulated lactating Holstein cows with varying body condition score (10 cows per

660

group were superovulated). Body condition

No. CL

score category

No. SO with

Total ova and

Transferrable

No.

No.

No.

zero response

embryos (%)

embryos (%)

morula

blastocysts

UFO/DGM

BCSC1

7.6 ± 1.6a

2

5.3 ± 1.6 (69.7)a

3.9 ± 0.6 (73.6)a

3.0 ± 0.4ab

1.0 ± 0.4a

1.4 ± 0.6

BCSC2

11.0 ± 1.6b

1

9.1 ± 1.6 (73.6)b

7.3 ± 0.8 (80.2)b

3.7 ± 0.6b

3.6 ± 0.6c

2.8 ± 0.4

BCSC3

10.8 ± 1.2b

1

9.5 ± 1.2 (87.0)c

7.9 ± 0.7 (83.2)b

5.5 ± 0.6c

2.4 ± 0.5b

1.6 ± 0.3

BCSC4

10.6 ± 1.0b

2

8.9 ± 1.0 (83.0)bc

6.5 ± 0.5 (73.0)a

3.1 ± 0.6b

3.4 ± 0.3bc

2.4 ± 0.2

BCSC5

6.1 ± 1.7a

4

4.8 ± 1.7 (75.4)a

3.3 ± 0.6 (68.8)a

1.8 ± 0.5a

2.5 ± 0.4a

1.6 ± 0.4

661

a-c

662

BCSC - Body condition score (1 to 5; 1, emaciated; 5, obese); category BCSC1, 2.00 to < 2.50; BCSC2, 2.50 to < 3.00; BCSC3, 3.00

663

to <3.50; BCSC4, 3.50 to <4.00; and BCSC5, 4.00 to 5.00; CL - Corpus lutea; SO - Superovulation; UFO - Unfertilized oocytes;

664

DGM - Degenerate embryos;

665

Total ova and embryos (%) = Total ova and embryos/ Number of CL;

666

Transferrable embryos (%) = Number of transferrable embryos/ Number of total embryos;

Within a column, means without a common superscript differed (P<0.05).

31

667

Table 2. Mean ± SEM reproductive data in supervoulated cows with varying %PMN in endometrial cytology. %PMN

No.

No. CL

flushes

No. SO with

Total ova and

Transferrable

No.

No.

No.

zero response

embryos (%)

embryos (%)

morula

blastocysts

UFO/DGM

0

26

10.7 ± 2.5a

2

8.9 ± 1.5 (83.2)a

7.4 ± 0.7 (83.1)a

4.1 ± 0.3a

3.3 ± 0.5a

1.5 ± 0.8

1 to 6

13

8.9 ± 1.4ab

2

7.1 ± 1.3 (79.8)b

5.4 ± 0.9 (76.1)b

3.4 ± 0.4a

2.0 ± 0.5b

1.7 ± 0.9

>6

11

6.9 ± 1.0b

6

4.8 ± 1.2 (69.6)c

2.5 ± 0.4 (47.9)c

1.6 ± 0.5b

0.9 ± 0.4b

2.3 ± 1.4

668

a,b

669

PMN - Polymorphonuclear neutrophil; CL - Corpus lutea; SO - Superovulation; UFO - Unfertilized oocytes; DGM - Degenerate

670

embryos;

671

Total ova and embryos (%) = Total ova and embryos / Number of CL;

672

Transferrable embryos (%) = Number of transferrable embryos/ Number of total embryos;

Within a column, means without a common superscript differed (P<0.05).

32

673 674 675

Fig. 1. Schematic overview of superovulation and embryo collection protocol. Briefly, on Day 0, embryo donor cows received a progesterone releasing vaginal insert

676

(CIDR; 1.38 g of progesterone; Eazi-Breed™ CIDR® Cattle Insert; Zoetis Animal Health, New

677

York, NY, USA) along with gonadorelin hydrochloride (GnRH; 2 mL (100 µg), im, Factrel®;

678

Zoetis Animal Health). On Day 4 (84 h after GnRH), superovulation with Folltropin-V®

679

(Follicle stimulating hormone (FSH) equivalent to 400 mg NIH-FSH-P1; im; Bioniche Animal

680

Health, Athens, GA, USA) was initiated, twice daily decreasing doses over 4.5 d. Luteolytic dose

681

of dinoprost (PGF2α; 5 mL (25 mg) im; Lutalyse® sterile solution; Zoetis Animal Health) was

682

administered with the last two FSH injections and progesterone inserts were removed with the

683

second last FSH injection, on Day 7 PM. Ovulation was induced with GnRH (100 µg im; Zoetis

684

Animal Health) 44 h after progesterone insert removal and donors bred by fixed-time artificially

685

insemination, 12 and 24 h later.

33

686 687

Fig. 2. Association of mean blood urea nitrogen (BUN) concentration (ng/mL), body condition

688

score and transferable embryo (%) of lactating Holstein cows with varying body condition

689

scores.1

690

Transferrable embryo (%) = Number of transferrable embryo/ Number of total embryo;

691

1

692

BCSC2, 2.50 to < 3.00; BCSC3, 3.00 to <3.50; BCSC4, 3.50 to <4.00; and BCSC5, 4.00 to 5.00;

693

A linear association between body condition score and BUN concentration (r2=0.82; P<0.05) and

694

a quadratic relationship between BUN concentrations and % transferrable embryos (r2=0.86;

695

P<0.05) was observed.

Body condition score (1 to 9; 1, emaciated; 9, obese) category (BCSC); BCSC1, 2.00 to < 2.50;

34

696 697

Fig. 3. Mean ± SEM serum concentrations of metabolic biomarkers in superovulated lactating

698

Holstein cows with various body condition categories.1

699

a,b

700

(P<0.05);

701

IGF - Insulin like growth factor; IL - Interleukin; TNF - Tumor necrosis factor;

702

1

703

2.50 to < 3.00 (BCSC2), 3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00

704

(BCSC5); Thin, BCSC1; Moderate to good, BCSC2, BCSC3 and BCSC4; Obese, BCSC5;

Within biomarker, means without a common superscript differed body condition categories

Body condition score (1 to 9; 1, emaciated; 9, obese) category (BCSC); 2.00 to < 2.50 (BCSC1),

35

705 706

Fig. 4. Mean ± SEM serum concentrations of metabolic biomarkers in superovulated lactating

707

Holstein cows with (≤ 6%PMN) or without (> 6%PMN) subclinical endometritis.

708

a,b

709

IGF - Insulin like growth factor; IL – Interleukin; TNF – Tumor necrosis factor;

Within biomarker, means without a common superscript differed (P<0.05);

36

Highlights •

Mean number of total ova and embryos was significantly higher for cows with no subclinical endometritis

•

Mean number of total ova and embryos was significantly lower for cows with BCS 2.00 to <2.50 and with BCS 4.00 to 5.00

•

Circulating adiponectin, leptin, insulin, IGF1 and TNFα were greater in cows with moderate to good body condition

•

Circulating adiponectin, leptin, IGF1 and insulin were greater in cows with no subclinical endometritis and TNFα and IL1β and IL6 were greater in cows with subclinical endometritis

•

Quadratic associations was observed between metabolic biomarkers, body condition, uterine PMN and superovulatory response