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Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes
Accepted Manuscript Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes S. Nandi, S.K. Tripathi, P.S.P. Gupta, S. Mondal PII:
S0093-691X(17)30351-5
DOI:
10.1016/j.theriogenology.2017.07.024
Reference:
THE 14190
To appear in:
Theriogenology
Received Date: 6 April 2017 Revised Date:
19 July 2017
Accepted Date: 20 July 2017
Please cite this article as: Nandi S, Tripathi SK, Gupta PSP, Mondal S, Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.07.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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Effect of metabolic stressors on survival and growth of in vitro cultured
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ovine preantral follicles and enclosed oocytes
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S. Nandi*, S.K.Tripathi, P.S.P.Gupta, S. Mondal
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ICAR-National Institute of Animal Nutrition and Physiology, Bangalore-560030, India
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*Corresponding author: ICAR-National Institute of Animal Nutrition and Physiology (ICARNIANP), Bangalore 560030, India; Email: [email protected] Abstract
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The present study was undertaken to study the effect of metabolic stressors like
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elevated levels of ammonia, urea, Non-esterified fatty acid (NEFA) and β-hydroxybutyric
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acid (BHB) on preantral follicle growth, survival, growth rates of oocytes enclosed in
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preantral follicles (PFs), maturation rates of oocytes recovered from cultured follicles,
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hormone production (estrogen and progesterone), reactive oxygen species (ROS) as well as
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superoxide dismutase (SOD) activity. Small pre-antral follicles (SPFs, 100– 250 µm) and
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large pre-antral follicles (LPFs, 250–450 µm) were isolated from slaughterhouse ovaries by
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a mechanical cum enzymatic method. SPFs and LPFs were cultured in vitro for 14 and 7
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days respectively and examined for their growth, survival and growth rates of enclosed
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oocytes in PFs exposed with different concentration of ammonia (0, 100, 150, 200, 250, 300
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and 400 µM), urea (0, 4, 4.5, 5, 5.5,6, 7 and 8 mM), NEFA [Basal NEFA (70µM): stearic
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acid, SA (10µM)+Palmitic acid, PA(20µM)+oleic acid, OA(40 µM), b) Medium combo (140
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µM): SA (20µM)+ PA(40 µM)+ OA(80 µM), c) High combo (210µM): SA (30 µM)+PA(60
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µM)+OA(120 µM), d) Very high Combo (280µM): SA(40µM)+PA(80µM)+OA(160µM)]
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and BHB (0, 0.5, 0.75, and 1µM). Results indicated that ammonia, urea, NEFA and BHB
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caused inhibition of survival and growth of in vitro cultured ovine PFs and enclosed oocytes
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at the levels of 300 µM, 8mM, high combo level of NEFA and 0.75 µM respectively. Our
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study may contribute to the identification of the mechanisms involved in decline of fertility
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due to metabolic and nutritional stress in ruminants.
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Key words: Preantral follicle, oocyte, growth, survival, metabolic stressors, ewes.
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ACCEPTED MANUSCRIPT Introduction
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The metabolites of protein digestion (ammonia and urea) affected different components of
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reproductive system of the ruminants as it was reported that ammonia affected the oocyte
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quality before ovulation, while urea mainly interfered negatively after fertilization [1].
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Similarly, energy deficit diet led to lipolysis which was characterized by elevated non-
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esterified fatty acid (NEFA) and β-hydroxybutyric acid (BHB) along with low glucose
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concentrations in serum [2]. These nutrient metabolites affected endocrine signalling and the
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quality of the oocyte and/or embryo [3]. Moreover, during the negative energy balance
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(NEB) accumulation of NEFA (derived from adipose tissue) in follicular fluid hampered the
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proliferation of the granulosa cells and thus jeopardized the oocyte development [4]. We
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earlier reported that the total NEFA, BHB, ammonia and urea above the physiological
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concentrations in serum and follicular fluids had been considered as nutritional and metabolic
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stressors [5, 6]. We had also shown that elevated ammonia and NEFA concentrations in the
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final maturation phase of oocytes in vitro were unfavourable for the oocytes developmental
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competence and subsequent embryo development [7, 8].
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It was never examined if or to what extent folliculogenesis, follicle quality, and the
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maturational competence of the enclosed oocyte in the preantral follicles (PFs) were affected
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by continuous exposure to elevated concentrations of ammonia, urea, NEFA, and BHB
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during follicle growth in vitro. Hence, the aim of this present study was to investigate the
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effects of exposure to elevated ammonia, urea, NEFA, and BHB concentrations on the
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frequency of follicular growth [both small preantral follicle (SPFs) and large preantral
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follicles (LPFs)], survival, oocyte maturation, reactive oxygen species (ROS) production,
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super oxide dismutase (SOD) enzyme activity and estrogen/ progesterone production during
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in vitro culture of PFs in ovine model.
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Material and method
Unless otherwise stated, culture media and chemicals were purchased from Sigma
Chemicals (St Louis, MO, USA).
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Ovaries
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Ovaries from mature, healthy, non-pregnant sheep (Ovis aries, age: 2-2.5 years) were
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collected from a local abattoir during breeding seasons (March to April, June to July and
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from September to October). Ovaries were brought to the laboratory in warm (32–35 0C) 2
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normal saline (0.9% NaCl) containing 50 µg ⁄ mL gentamicin sulfate within one hour of
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slaughter.
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Recovery and culture of pre-antral follicles
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to isolate PFs from sheep ovaries [9]. PFs with normal follicular outline, compact granulosa
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cells and visible oocyte were only selected. PFs were classified according to the criteria
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described earlier [9, 10]. The diameter of SPFs, Fig 1 ranged from 100 to 250 µm and had 2-4
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layers of granulosa cells. The diameter of LPFs, Fig 2 ranged from 250 to 450 µm and had
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more than 4 layers of granulosa cells although they occasionally processed small antral cavity
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[10].The isolated PFs (SPFs and LPFs) were washed in the isolation and washing medium
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containing minimum essential medium (MEM) supplemented with bovine serum albumin
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(BSA, 0.3%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and
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gentamicin (50µg ⁄ mL).
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A combined mechanical and enzymatic method developed in our laboratory was used
Only viable PFs as assessed by trypan blue staining method [11] were used for
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culture. In brief, PFs were incubated in 0.04% trypan blue for 2 min at room temperature. PFs
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stained with trypan blue were considered as dead (Fig. 3) and unstained PFs were considered
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as alive (Fig. 4).The isolated PFs (two to three in a group) were transferred in 100µL droplets
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of the culture medium under paraffin oil in a 35-mm petri dish and cultured in a CO2
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incubator (38.5°C, 5% CO2 in air, 90–95% relative humidity) for 7 days (LPFs) or 14 days
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(SPFs). The control culture medium was MEM supplemented with BSA (0.3%), glutamine (2
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mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM), insulin–selenium–transferin (1%)
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and gentamicin (50 µg ⁄ ml) and FSH-P (7µg ⁄ ml; biological potency = 7 U⁄ mg; F2293; LH
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≤1%). The first day of culture was designated as Day-0. The medium was replenished at the
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end of Day-2 and Day-4 for 7 day culture and additionally at the end of Day-6, Day-8, Day-
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10 and Day-12 for a 14 day culture.
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Follicle diameters (µm) were assessed by measuring the distance between 2 sides of
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the follicle, straight through the center of the oocyte using an eye piece micrometer fitted on
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the stero zoom microscope (magnification X200) [12]. Oocyte diameters included zona-
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pellucida thickness as it was reported that the formation of the zona pellucida always
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occurred during the PF stage [13].The final diameter of the follicles was recorded together
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with the presence and absence of an antral cavity (a visible translucent area within the
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granulosa cell mass (Fig. 5). The growth rate measured was the cumulative growth rate.
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ACCEPTED MANUSCRIPT The growth, survival rates of PFs and growth rate of oocytes in enclosed SPFs were
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calculated as described earlier [9]:
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of follicle] ⁄ seven (LPFs) or fourteen (SPFs).
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Survival rate (%) = [No. of viable follicles at the end of culture) ⁄ no of viable follicles put into culture] ×100.
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Growth rate of follicle (µm⁄ day) = [Final diameter (µm) of follicle - Initial diameter (µm)
3. Growth rate of oocyte (µm ⁄ day) = [Final diameter (µm) of oocyte - Initial diameter (µm) of oocyte] ⁄ seven (LPFs) or fourteen (SPFs). Oocyte recovery and maturation
At the end of the culture, all of the healthy SPFs and LPFs were cautiously opened
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mechanically using (26G) hypodermic needles attached to a 2mL syringe barrels under a
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stereo zoom microscope. Oocytes with a homogeneous cytoplasm and surrounded by at least
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two layer of granulosa cells were selected for in vitro maturation (IVM). IVM was performed
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as described earlier [14]. In brief, oocytes were washed three times in washing medium
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(TCM 199 + 10% FBS, 50µg/ml gentamicin) and then twice in the maturation medium
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(TCM-199 + FBS (10%) + Follicle Stimulating Hormone-ovine (FSH-O, 10 µg/ml) + 50
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µg/ml gentamicin). Oocytes (6-8) were matured in 50µL maturation medium droplets of
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TCM-199 + FBS (10%) + Follicle Stimulating Hormone-ovine (FSH-O, 10 µg/ml) + 50
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µg/ml gentamicin under oil in a 35-mm Petri dish in a CO2 incubator (38.5◦C, 5% CO2 in air,
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90%–95% relative humidity) for 24 h. The mineral oil was pre-equilibrated with the culture
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medium. Oocytes with an expanded cumulus cell mass (Fig 6) and with an extruded first
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polar body in the perivitelline space (Fig 7) were considered as matured [15].
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Levels of ROS
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Levels of ROS were determined for the culture media by the spectrofluorimetric
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method, using the 20, 70-dichlorofluorescein diacetate (DCF-D) assay [16]. The medium was
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incubated with 5mL of DCF-D (1 mM), and the oxidation of DCF-D to fluorescente
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dichlorofluorescein was measured for ROS detection. Dichlorofluorescein fluorescence
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intensity emission was recorded at 520nm (with 480 nm excitation) 120 minutes after the
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addition of DCHFDA to the medium.
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Hormone assay
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The spent medium obtained at the end of Day-7 and Day-14 for LPFs and SPFs
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respectively were pooled per plate and stored at -80 ºC for hormonal assays. E2 and P4
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concentrations were determined by ELIAS kits (commercial kits for clinical use in humans, 4
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Diagnostics Biochemical’s Pvt Inc., Ontario, Canada). The calibrator ranges were 20 to 3200
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pg/mL and 0.15 to 20 ng/mL for E2 and P4 respectively. Twenty five microlitres of samples
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were used in quadruplicate. All samples were run in one assay to avoid inter-assay variation
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and intra-assay variations were 2.4 and 2.7% for E2 and P4 respectively.
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Assessment of SOD activity Twenty PFs from each treatment group were collected and used on the same day for
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SOD activity assay. Enzymatic extracts of in vitro cultured SPFs and LPFs at the end of final
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culture period (i.e 7 days for SPFs and 14 for LPFs) were obtained by homogenizing then in
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cold 20 mM HEPES buffer followed by centrifugation for 5 min at 4 °C. Total SOD activity
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(inhibition rate %) was determined by the SOD Assay Kit-WST (Sigma-aldrich, USA)
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according to the manufacturer’s recommendations. The assay was replicated three times. The
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absorbance read at 450nm with a micro plate reader.
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Selection of levels of metabolic stressors
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The types and concentrations of metabolic stressors used in this study were based on
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earlier in vivo and in vitro studies [5-8, 14, 15] in our laboratory. In brief, the mean follicular
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fluid ammonia, urea, NEFA and BHB levels were 132 Vs 157µM; 4.0 Vs 6.0 mM; 78.0 Vs
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99.0µM; and 0.5 Vs 0.72mM in post parturient and high protein diet scenario (metabolically
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stressed ewes) respectively. The ammonia level ranged from 94 to 412 µM, urea from 3 to
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8.6mM, NEFA from 68 to 256 µM and BHB from 0.42 to 0.94 mM in metabolic stressed
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ewes. We also reported that the mean basal NEFA level in ewe follicular fluid was 70.4 µM,
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and the oleic, palmitic and stearic acids were the three predominant free fatty acids in
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follicular fluid and the average relative presences of these NEFAs were 40%, 25% and 15%
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respectively. We used the following composition of NEFA in the present study: a) Basal
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NEFA (70µM): stearic acid, SA (10µM)+Palmitic acid, PA(20µM)+oleic acid, OA(40 µM),
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b) Medium combo (140 µM): SA (20µM)+ PA(40µM)+ OA(80 µM), c) High combo (210
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µM): SA (30µM)+PA(60µM)+OA(120µM), d) Very high Combo (280µM): SA(40
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µM)+PA(80µM)+OA(160µM).
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Experiments
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Four different experiments, to investigate the influence of inclusion of different
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concentration of ammonia [0 (control), 100 (Basal level), 150, 200, 250, 300 and 400µM],
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urea [0 (control), 4 (basal level), 4.5, 5, 5.5,6, 7 and 8mM], NEFA [0 (control), Basal NEFA,
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High-Combo and Very high-combo] and BHB [0(control), 0.5 (basal), 0.75, and 1µM] on the 5
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in vitro development of ovine PFs were conducted. Each of the experiments was replicated
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for 8 times each for SPFs and LPFs. Each replicate consisted of twelve 100µL droplets with
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2-3 PFs for each treatment.
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Statistical analysis
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by a prospective, randomized study. The interactions between follicle diameter and treatment
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as well as replicate and treatment were performed as per the model described earlier [12]
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however as we did not get any significant interaction, we omitted from the final analysis.
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Significant differences in experiments between the growth rates of PFs and oocytes, survival
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rates of PFs, maturation rate of oocytes, hormone production and SOD activity were analyzed
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by ANOVA and the respective means were compared using Tukey’s test where treatment
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was entered as a fixed factor, and replicate as a random factor. The differences between the
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mean values of all parameters were tested by t-test wherever the comparison was made
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between SPFs and LPFs. The percentage values were transformed by arcsine square root
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before analysis. Difference was considered to be significant at p< 0.05. The computer assisted
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statistical software package (Graph Pad Prism, San Diego, CA, USA) was used for analyzing
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the data.
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Results
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The effects of each factor on the follicular features (growth ⁄ survival) were evaluated
The effects of ammonia, urea, NEFA and BHB on in vitro development of SPFs and
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LPFs are presented in Tables 1-4. In all the four experiments LPFs exhibited better frequency
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of survival, rates of growth and maturation of oocytes to MII stage as well as estrogen
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production (Tables 1-4). The frequency of survival, rate of growth of PFs' and maturation of
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oocytes to MII stage began to decrease significantly on exposure to 300 µM ammonia and
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was further decreased at 400µM in both SPFs’ and LPFs’(Table 1). While the estrogen
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production was reduced at 400µM of ammonia, SOD activity started to decline significantly
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at 200µM concentration (Table1).However, the progesterone production was not adversely
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affected by exposure to ammonia (Table 1).
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The frequency of survival, rate of growth of PFs' and maturation of oocytes to MII,
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estrogen production and SOD activity began to decrease significantly on exposure to 8mM of
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urea in both SPFs’ and LPFs’(Table 2). However, the progesterone production was not
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adversely affected by exposure to urea (Table 2).
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stage began to decrease significantly on exposure to High combo NEFA and was further
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decreased at Very high combo NEFA in both SPFs’ and LPFs’ (Table 3), while the estrogen
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production was reduced at Medium combo NEFA compared with Basal NEFA (Table 3).
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Further, significant reduction in estrogen production was observed when LPFs were treated
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with high level of NEFA compared to medium level NEFA, SOD activity started to decline
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significantly at Medium combo NEFA concentration and was maximum at very high combo
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NEFA in both SPFs’ and LPFs’ (Table 3). However, the progesterone production was not
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adversely affected by exposure to ammonia (Table 3).
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The rate of growth of LPFs and survival rate of both SPFs and LPFs were
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significantly decreased on exposure to 0.75µM BHB compared to lower levels (Table 4).
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Further increment of BHB level to 1µM level decreased the survival rate of only LPFs. The
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rate of growth and maturation of oocytes to MII stage began to decrease significantly on
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exposure to 1.0µM BHB concentration (Table 4), while SOD activity started to decline
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significantly at 0.75µM concentration and was maximum at 1.00µM in both SPFs’ and LPFs’
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(Table4). However, the estrogen and progesterone production was not adversely affected by
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exposure to BHB (Table 4).
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Discussion
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The impact of metabolic stress on the preantral phase of folliculogenesis in domestic
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animals is unknown. Therefore, we studied the effect of metabolic stressors on PFs
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(representing 90% of ovarian follicle population) using ovine follicles cultured in vitro.
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During the journey of PFs to ovulation, follicles might be under different stresses (heat stress,
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nutritional stress, disease or other factors) that could impair the developmental competence of
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follicle-oocyte complexes [17]. Similarly, primary follicles exposed to stress were less
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competent to produce ample quantities of estrogens and progesterone and such follicles
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contained poor quality oocytes [18].
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It was reported that exposure of bovine oocytes to ammonia concentrations of 29 to
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356mM during IVM did not adversely influence oocyte development which indicated that
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bovine oocytes could tolerate elevated concentrations of ammonia during IVM as determined
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by the subsequent embryonic development in vitro [19]. In contrast, we observed that
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maturation rates of oocytes retrieved from cultured PFs’ in the present study as well as those
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retrieved from antral follicles in an earlier study [16] were significantly lowered when
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exposed to 250µM ammonia. Similarly, it was reported that the high urea level of serum
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resulted in penetration of urea into follicles; this did not impair the development of such
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follicles [20]. However, our results suggested that PF growth was impaired when exposed to
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8 mM level of urea. Long-term exposure of elevated NEFA concentration (720µM) to murine follicles
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was reported to influence the follicular growth, with the most marked effect induced by the
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high stearic acid (280µM) treatment [12]. The same authors also reported that oocytes
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originated from the NEFA-exposed follicles showed notably low oocyte developmental
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competence. High NEFA also reported to cause amended glucose metabolism and increased
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beta-oxidation, and its outcome was higher concentration of ROS production [21]. Similarly
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exposure of bovine oocyte to high concentration of NEFA (425µM) was reported to affect β-
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oxidation or lipogenesis and vital mechanisms for developmental competence of oocytes
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[22]. It was reported that oocytes matured under high saturated NEFA environment showed
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significantly distorted metabolic pathways at both gene transcription and gene function levels
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[12]. We observed significant changes in terms of growth and survival of PFs when cultured
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in medium containing high combo NEFA. In mice, elevated NEFA concentration negatively
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affected pathways involved in apoptosis, lipid metabolism, oxidative stress and
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steroidogenesis, which was confirmed by P4, oestradiol and inhibin B analyses in spent
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medium [12]. Valckx and her co-workers [23] cultured murine early secondary PFs up to Day
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13, followed by oocyte isolation, fertilisation and embryo culture. When these follicles are
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exposed to elevated NEFA concentrations (720µM) throughout follicle growth or only during
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the final maturation phase, long-term exposure (13 days) severely impaired oocyte
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developmental competence when compared with short-term NEFA exposure. This
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strengthened our observation that not only final oocyte maturation, but also the preceding
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period of follicular and oocyte growth were influenced by metabolic stressors. It should also
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be noted that simple stomached animals could have lower tolerance for some of these
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metabolites; ruminants might be less sensitive since these metabolites were normally
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produced in significant quantities during digestion in rumen and passed into circulation.
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Ketone bodies like BHB etc were negatively associated with fertility (disturbances in
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LH pulse frequency, low growth of the follicle, and decreased progesterone and estradiol
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secretions) [24]. Our findings were in accordance to the earlier report [25] wherein it was
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observed that the very high BHB concentrations as well as the concomitant hypoglycaemic
32
conditions seemed to be responsible for adverse effect on development competence of
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oocyte. It was also reported that in bovine more than 50mg/l concentration of BHB in
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follicular fluid had been negatively correlated with the percentage of good-quality oocytes 8
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and their developmental potential [26]. This was in agreement with our findings wherein the
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SPFs and LPFs exposed to high BHB concentration showed less development and survival
3
rates. In an earlier study it was reported that there was a reduction in progesterone
5
production and no change of estrogen production under high NEFA in early PFs was
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recorded [12]. However we observed no change in progesterone and reduction in estrogen
7
production under metabolic stress. Less estrogen production in the present study might be
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attributed to low aromatase activity due to metabolic stressors as examined by ELISA (Nandi
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S, unpublished data) or by low expression of caveolin 1, a regulator of aromatase activity [27].
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The detrimental effects of the stress on PFs might also be due to generation of high
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concentration of ROS that affected the growth of PFs as well as oocytes [16]. Moreover, the
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metabolic stress might led to higher expression or upregulation of proapoptotic genes and
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cellular apoptosis as reported earlier [28, 29]. The increased concentration of metabolic
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stressors in all the tested treatments in the present study caused a decline in follicle viability
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and growth, signifying that this incident might possibly arbitrated by ROS.
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Our results differed from a possibility as suggested earlier that oocytes within PFs
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were completely insensitive to the metabolic stressors [30]. The same authors reported that
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the oocytes were exposed to a short period of time in in-vitro system whereas in in-vivo the
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oocytes were exposed to these concentrations for several days or even weeks. According to
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the same authors in the ideal model, PFs should be cultured in metabolic stressors for several
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weeks.
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Subsequent build-up of ammonium, generated from the breakdown of amino acids
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and glutamine in the culture medium, impaired cell growth [31, 32]. Ammonia concentrations
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in fresh IVM control medium were below detectable levels and increased from 4 to 7 mM at
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the end of the 2 days culture period (the media were replenished every 2 days) in the present
105
study. We had used medium devoid of glutamine and used sodium pyruvate as energy source.
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This might be the cause of lower amount of ammonia generated in the IVM medium in the
107
present study. We used periodic replacement of medium as the gradual increase in the culture
108
medium volume through the addition of fresh medium would provide new nutrients and also
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maintain the medium with substances produced in the different compartments of the follicle
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[33]. Periodic addition of medium (small supplementation method) was recommended
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because it was more practical, maintained survival and promoted the development of
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preantral follicles in vitro [34].
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ACCEPTED MANUSCRIPT The present study had shown, for the first time, ovarian follicles even at the smaller
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size were affected by changing milieu of metabolites. All the metabolic stressors (ammonia,
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urea, NEFA and BHB) impaired the functions of ovarian PFs and their enclosed oocytes at
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the physiological levels observed during metabolic stress conditions. Ammonia, urea, NEFA
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and BHB caused inhibition of survival and growth of in vitro cultured ovine PFs and
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enclosed oocytes at the levels of 300 µM, 8mM, high combo level of NEFA and 0.75 µM
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respectively The results of the present study might contribute to the identification of the
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mechanisms and factors involved in reduced fertility due to metabolic and nutritional stress in
9
ruminants. Acknowledgement
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We are grateful to the Director, NIANP, Bangalore for providing the necessary facilities. We thank Mr. Gyan Prakash for technical assistance. Financial help from the
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Department
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BT/PR7131/AAQ/1/526/2012) is gratefully acknowledged. We declare no conflicts of
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interests.
of
Biotechnology,
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India
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[2] Leroy JLMR, Vanholder T, Delanghe JR, Opsomer G, Van Soom A, Bols PEJ, Dewulf J,
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[6] Farman M, Tripathi SK, Nandi S, Girish KV. (2015). Follicular fluid concentrations of
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[7] Nandi S, Mondal S, Pal DT, Gupta PSP. Effect of ammonia-generating diet on ovine
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methods. Animal Reproduction Science 2015,161: 23–31.
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ACCEPTED MANUSCRIPT
ROS (fluorescence units) Estrogen (pg/mL) production Progesterone (ng/mL) production
77.5±3.5a
LPF SPF* LPF* SPF LPF SPF* LPF* SPF
81.2±3.9a 2.9±0.21a 11.6±0.34a 3.7±0.2 a 3.4±0.4 a 46.5±2.7a 62.6±3.6a 15.3 ±3.4 a
77.3±3.6a 2.8±0.21a 11.4±0.32a 3.6±0.3 a 3.4±0.5 a 44.3±1.8a 61.8±1.6a 15.8 ±3.1 a
77.3±3.3a 2.8±0.19a 9.8±0.16a 3.3±0.4 a 3.1±0.1 a 45.6±1.7a 60.6±3.2a 15.8 ±3.4 a
LPF SPF*
16.5 ±2.4 a 98.6±7.8a
15.9 ±3.0 a 94.3±6.0a
16.1 ±2.5 a 92.9±5.8a
15.8 ±2.7 a 93.1±5.4a
21.4 ±3.1 b 92.3±4.6a
22.1 ±2.2b 92.6±3.3a
26.7 ±2.9c 68.6±2.8b
LPF* SPF
148.4±9.8a 3.4±0.7a
148.0±8.2a 3.4±0.9a
149.8±7.6a 3.2±0.3a
128.4±4.5a 3.2±0.1a
128.7±6.2a 3.2±0.5a
120.1±4.3a 3.2±0.3 a
87.8±5.8b 3.2±0.7 a
LPF SPF
4.6±0.1a 56.2±1.2a
4.6±0.1a 55.4±0.9a
4.5±0.2a 49.3±1.5a
4.3±0.3a 38.4±2.3b
4.3±0.2a 36.1±1.6b
4.2±0.3a 20.4±0.3c
4.2±0.2a 18.1±0.8c
40.1±4.2b
38.4±3.8b
22.7±1.4c
16.6±1.7c
73.3±3.1a
200 (n=208)
250 (n=204)
300 (n=216)
400 (n=216)
74.5±2.5a
70.4±3.1a
62.7±2.4b
47.1±2.3c
76.5±2.3a 2.6±0.20a 9.1±0.23a 3.3±0.2 a 3.2±0.3 a 42.4±3.6a 58.2±2.0a 15.7 ±3.2 a
72.0±3.2a 2.6±0.22a 8.8±0.25a 2.8±0.3 a 2.9±0.4 a 30.3±1.9b 47.1±1.7b 20.3 ±2.5b
66.4±2.7b 2.1±0.17b 3.9±0.16b 1.3±0.3 b 1.4±0.6 b 28.6±0.8b 42.4±0.7b 21.6 ±3.2b
45.3±2.7c 1.3±0.12c 2.4±1.7c 0.7±0.2 c 0.9±0.3 b 15.7±1.5c 21.7±0.4c 25.4 ±2.5c
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150 (n=224)
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Growth (in enclosed follicles) rate of oocyte (µM/day) Maturation rate of Oocytes (%)
SPF
100-Basal value (n=192) 75.2±3.2a
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Growth rate of PFs (µM/day)
0-Control (n=192)
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Survival rate of PFs, %
Concentration of ammonia, µM
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Parameter studied
Follicle type
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Table 1: Effect of ammonia on small and large PFs cultured in vitro
SOD enzyme activity (Inhibition rate %) LPF 61.2±3.1a 60.1±2.5a 56.4±1.8a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
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Maturation rate of Oocytes (%) ROS (fluorescence units)
Estrogen (pg/mL) production
74.6±2.2a 77.3±2.9a 2.7±0.23a 10.5±0.21a 3.3±0.3a 3.7±0.9 a 42.8±2.7a
72.1±3.2a 74.2±3.1a 2.6±0.20a 9.4±0.20a 3.3±0.4a 3.4±0.3 a 40.5±2.5a
71.3±2.1a 74.3±2.6a 2.7±0.16a 9.1±0.19a 3.1±0.3a 3.4±0.2 a 40.2±3.2a
70.3±2.1a 74.4±2.4 a 2.7±0.10a 9.3±1.4a 3.0±0.3 a 3.2±0.4 a 38.6±3.4a
68.2±1.8a 70.2±2.7a 2.6±0.27a 9.03±1.5a 3.0±0.5a 3.1±0.1a 36.6±2.4a
54.3±2.4 b 52.3±1.4 b 2.1±0.21b 7.04±1.1b 1.9±0.8b 1.7±0.4b 20.5±3.2b
62.6±2.4a
60.3±3.8a
60.2±2.2a
60.4±2.1a
58.5±3.4a
37.1±2.3b
75.3±3.1a 77.2±3.5a 2.9±0.22a 10.9±0.31a 3.6±0.2a 4.1±0.6 a 44.8±2.6a
LPF*
66.4±2.4a
63.6±3.2a
SPF LPF
12.5 ±4.4 a 14.1 ±2.4 a
13.1 ±2.1 a 14.8 ±2.0 a
13.4 ±2.4 a 15.5 ±3.5 a
13.7 ±1.2 a 15.2 ±1.7 a
14.3 ±3.5a 16.4 ±3.2 a
14.8 ±2.2a 16.8 ±2.5a
22.4 ±4.5b 28.7 ±3.0b
26.1±2.3c 30.5±2.1c
SPF*
99.3±6.3a
98.7±5.2a
97.5±8.2a
98.3±5.9a
97.4±6.3a
97.1±6.6a
96.6±7.9a
75.3±4.8b
138.2±8. a 3.4±0.4 a 4.5±0.1a 52.6±2.8a 55.6±1.9a
135.7±5.3a 3.2±0.3 a 4.5±0.2a 51.2±3.7a 54.1±2.3a
132.9±6.9a 3.2±0.4 a 4.5±0.2a 51.8±3.2a 54.7±2.8a
133.2±6.1a 3.0±0.3a 4.4±0.2a 50.7±3.1a 52.5±2.2a
93.1±7.9b 3.1±0.2a 4.3±0.2a 43.8±2.5b 44.7±2.9b
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Growth (in enclosed follicles) rate of oocyte (µM/day)
8 (n=216)
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Growth rate of PFs (µM/day)
7 (n=204)
SPF LPF SPF* LPF* SPF LPF SPF*
EP
Survival rate of PFs, %
4.5 (n=204)
4-Basal value (n=204) 74.6±1.7a 76.3±3.0a 2.9±0.20a 10.7±0.34a 3.6±0.4a 4.2±0.7 a 43.4±2.9a
Parameter studied
0-Control (n=192)
Concentration of urea (mM) 5 5.5 6 (n=216) (n=216) (n=224)
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Table 2: Effect of urea on small and large PFs cultured in vitro
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LPF* 147.8±9.5a 147.3±8.6a 146.2±7.5a a a SPF 3.4±0.7 3.4±0.2 3.5±0.8 a Progesterone (ng/mL) production LPF 4.6±0.2a 4.6±0.3a 4.5±0.1a a a SPF 55.4±3.6 56.2±2.4 53.4±4.2a SOD enzyme activity (Inhibition rate %) LPF 60.7±1.9a 60.4±2.5a 56.1±1.6a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
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Table 3: Effect of NEFA on small and large PFs cultured in vitro
Parameter studied
Concentration of NEFA (µM) 0-Control (n=204)
Basal NEFA (n=204)
SPF 79.4±2.5a 73.5±2.2a a LPF 84.3±2.4 77.1±2.6a SPF* 2.9±0.28a 2.8±0.25a Growth rate (µM/day) a LPF* 10.9±0.29 10.7±0.31a 3.5±0.3a SPF 3.6±0.2a Growth (in enclosed follicles) rate of oocyte (µM/day) LPF 4.3±0.2a 4.2±0.3a a SPF* 41.3±1.9 42.5±2.5a Maturation rate of Oocytes (%) LPF* 63.5±2.3a 62.4±2.7a a SPF 8.2 ±3.4 8.5 ±23.1 a ROS (fluorescence units) LPF 11.4 ±2.1 a 11.9 ±1.0 a a 94.2±5.2a SPF* 94.6±3.2 Estrogen (pg/mL) production LPF* 140.0±8.6a 143.7±7.2a SPF 3.7±0.4a 3.6±1.2a Progesterone (ng/mL) a production LPF 4.4±0.1 4.4±0.8a SPF 59.3±2.2a 55.4±1.5a SOD enzyme activity a (Inhibition rate %) LPF 57.4±4.5 57.6±2.3a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
Medium Combo (n=208)
High Combo (n=216)
Very high Combo (n=224)
74.1±1.1a 79.4±1.4a 2.7±0.18a 9.1±0.9a 3.4±0.5a 3.9±0.3a 40.4±2.4a 60.6±3.6a 9.1 ±4.4 a 12.4 ±2.5 a 80.6±2.9b 117.7±9.3b 3.5±0.6a 4.3±0.3a 31.4±1.3b 37.6±1.6b
63.3±2.1b 69.6±2.9b 2.4±0.16b 4.4±0.24b 2.1±0.9b 2.4±0.4b 27.4±2.4b 40.7±2.6b 16.8 ±5.2 b 20.4 ±4.7 b 70.1±1.6b 80.3±6.2c 3.3±0.1a 4.3±0.5a 20.3±1.2c 22.4±2.2c
53.4±2.2c 54.4±2.7c 2.1±0.20c 4.3±0.22c 0.4±0.6c 0.9 ±0.2c 13.3±1.2c 18.5±1.4c 18.3 ±2.5b 22.4 ±2.2 b 68.8±2.6b 77.7±4.5c 3.2±0.1a 4.2±0.3a 18.7±1.3c 21.5±2.3c
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Survival rate of PFs, %
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Follicle type
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Table 4: Effect of BHB on small and large PFs cultured in vitro
Parameter studied
1.00 (n=222) 66.1±1.7b 69.6±0.9c 2.2±0.1a 3.2±1.6b 2.6±0.4 b 3.7±0.2 b 28.6±1.4 b 40.4±2.5b 16.7 ±2.2 b 20.2 ±2.5 b 95.1±5.1a 132.6±8.6a 3.6±0.09a 4.0±0.1 a 36. ±2.4c 20.1±3.4c
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0-Control (n=208) SPF 80.1±2.4a Survival rate of PFs, % LPF 83.3±1.4a SPF* 2.8±0.4a Growth rate (µM/day) LPF* 10.6±2.1a SPF 3.5±0.4 a Growth (in enclosed follicles) rate of oocyte (µM/day LPF 4.0±0.6 a SPF* 43.6±2.7a Maturation rate of Oocytes (%) LPF* 62.6±2.6a SPF 10.8 ±1.9 a ROS (fluorescence units) LPF 12.1 ±1.4 a SPF* 96.2±4.2a Estrogen (pg/mL) production LPF* 142.3±9.4a SPF 3.9±0.1a Progesterone (ng/mL) production LPF 4.2±0.1a SPF 60.2±2.5a SOD enzyme activity (Inhibition rate %) LPF 63.1±3.6a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
Concentration of BHB (µM) 0.5-Basal value 0.75 (n=216) (n=218) 82.4±3.7a 70.6±3.6 b 81..1±0.6a 75.4±1.4b 2.7±0.6a 2.4±0.3a a 9.9±1.7 6.4±1.4b 3.4±0.6 a 3.2±0.5 a 4.1±0.3 a 4.3±0.4 a a 41.6±2.7 40.4±2.5a 62.7±2.5a 58.2±3.4a a 11.4 ±2.8 15.4 ±4.4 b 12.8 ±2.5 a 18.7 ±3.9 b a 95.7±2.5 94.4±3.8a 139.3±8.4a 135.5±7.5a a 3.7±0.2 3.8±0.1a 4.0±0.2a 4.1±0.1a a 57.4±1.6 40.7±1.9b 59.3±2.9a 34.2±2.4b
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Follicle type
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Fig. 1. Small Preantral follicle
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Fig. 2. Large preantral follicle
Fig. 4. Preantral follicle (alive)
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Fig. 3. Preantral follicles (Dead)
Fig. 5. Follicle with antrum
Fig 6. Oocytes with expanded cumulus cell mass
Fig 7: Oocyte with extruded first polar body
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Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes S. Nandi*, S.K.Tripathi, P.S.P.Gupta, S. Mondal
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ICAR-National Institute of Animal Nutrition and Physiology, Bangalore-560030, India
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*Corresponding author: ICAR-National Institute of Animal Nutrition and Physiology (ICARNIANP), Bangalore 560030, India; Email: [email protected]
Highlights
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Ovarian follicles even at the smaller size were affected by changing milieu of metabolites in ovarian tissues and follicular fluid. Increased levels of ammonia, urea, NEFA and BHB impaired the functions of ovarian preantral follicles and their enclosed oocytes
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S0093-691X(17)30351-5
DOI:
10.1016/j.theriogenology.2017.07.024
Reference:
THE 14190
To appear in:
Theriogenology
Received Date: 6 April 2017 Revised Date:
19 July 2017
Accepted Date: 20 July 2017
Please cite this article as: Nandi S, Tripathi SK, Gupta PSP, Mondal S, Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.07.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT
REVISED
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Effect of metabolic stressors on survival and growth of in vitro cultured
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ovine preantral follicles and enclosed oocytes
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S. Nandi*, S.K.Tripathi, P.S.P.Gupta, S. Mondal
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ICAR-National Institute of Animal Nutrition and Physiology, Bangalore-560030, India
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*Corresponding author: ICAR-National Institute of Animal Nutrition and Physiology (ICARNIANP), Bangalore 560030, India; Email: [email protected] Abstract
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The present study was undertaken to study the effect of metabolic stressors like
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elevated levels of ammonia, urea, Non-esterified fatty acid (NEFA) and β-hydroxybutyric
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acid (BHB) on preantral follicle growth, survival, growth rates of oocytes enclosed in
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preantral follicles (PFs), maturation rates of oocytes recovered from cultured follicles,
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hormone production (estrogen and progesterone), reactive oxygen species (ROS) as well as
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superoxide dismutase (SOD) activity. Small pre-antral follicles (SPFs, 100– 250 µm) and
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large pre-antral follicles (LPFs, 250–450 µm) were isolated from slaughterhouse ovaries by
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a mechanical cum enzymatic method. SPFs and LPFs were cultured in vitro for 14 and 7
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days respectively and examined for their growth, survival and growth rates of enclosed
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oocytes in PFs exposed with different concentration of ammonia (0, 100, 150, 200, 250, 300
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and 400 µM), urea (0, 4, 4.5, 5, 5.5,6, 7 and 8 mM), NEFA [Basal NEFA (70µM): stearic
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acid, SA (10µM)+Palmitic acid, PA(20µM)+oleic acid, OA(40 µM), b) Medium combo (140
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µM): SA (20µM)+ PA(40 µM)+ OA(80 µM), c) High combo (210µM): SA (30 µM)+PA(60
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µM)+OA(120 µM), d) Very high Combo (280µM): SA(40µM)+PA(80µM)+OA(160µM)]
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and BHB (0, 0.5, 0.75, and 1µM). Results indicated that ammonia, urea, NEFA and BHB
26
caused inhibition of survival and growth of in vitro cultured ovine PFs and enclosed oocytes
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at the levels of 300 µM, 8mM, high combo level of NEFA and 0.75 µM respectively. Our
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study may contribute to the identification of the mechanisms involved in decline of fertility
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due to metabolic and nutritional stress in ruminants.
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Key words: Preantral follicle, oocyte, growth, survival, metabolic stressors, ewes.
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ACCEPTED MANUSCRIPT Introduction
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The metabolites of protein digestion (ammonia and urea) affected different components of
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reproductive system of the ruminants as it was reported that ammonia affected the oocyte
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quality before ovulation, while urea mainly interfered negatively after fertilization [1].
5
Similarly, energy deficit diet led to lipolysis which was characterized by elevated non-
6
esterified fatty acid (NEFA) and β-hydroxybutyric acid (BHB) along with low glucose
7
concentrations in serum [2]. These nutrient metabolites affected endocrine signalling and the
8
quality of the oocyte and/or embryo [3]. Moreover, during the negative energy balance
9
(NEB) accumulation of NEFA (derived from adipose tissue) in follicular fluid hampered the
10
proliferation of the granulosa cells and thus jeopardized the oocyte development [4]. We
11
earlier reported that the total NEFA, BHB, ammonia and urea above the physiological
12
concentrations in serum and follicular fluids had been considered as nutritional and metabolic
13
stressors [5, 6]. We had also shown that elevated ammonia and NEFA concentrations in the
14
final maturation phase of oocytes in vitro were unfavourable for the oocytes developmental
15
competence and subsequent embryo development [7, 8].
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It was never examined if or to what extent folliculogenesis, follicle quality, and the
17
maturational competence of the enclosed oocyte in the preantral follicles (PFs) were affected
18
by continuous exposure to elevated concentrations of ammonia, urea, NEFA, and BHB
19
during follicle growth in vitro. Hence, the aim of this present study was to investigate the
20
effects of exposure to elevated ammonia, urea, NEFA, and BHB concentrations on the
21
frequency of follicular growth [both small preantral follicle (SPFs) and large preantral
22
follicles (LPFs)], survival, oocyte maturation, reactive oxygen species (ROS) production,
23
super oxide dismutase (SOD) enzyme activity and estrogen/ progesterone production during
24
in vitro culture of PFs in ovine model.
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Material and method
Unless otherwise stated, culture media and chemicals were purchased from Sigma
Chemicals (St Louis, MO, USA).
29 30
Ovaries
31
Ovaries from mature, healthy, non-pregnant sheep (Ovis aries, age: 2-2.5 years) were
32
collected from a local abattoir during breeding seasons (March to April, June to July and
33
from September to October). Ovaries were brought to the laboratory in warm (32–35 0C) 2
ACCEPTED MANUSCRIPT 1
normal saline (0.9% NaCl) containing 50 µg ⁄ mL gentamicin sulfate within one hour of
2
slaughter.
3 4 5
Recovery and culture of pre-antral follicles
6
to isolate PFs from sheep ovaries [9]. PFs with normal follicular outline, compact granulosa
7
cells and visible oocyte were only selected. PFs were classified according to the criteria
8
described earlier [9, 10]. The diameter of SPFs, Fig 1 ranged from 100 to 250 µm and had 2-4
9
layers of granulosa cells. The diameter of LPFs, Fig 2 ranged from 250 to 450 µm and had
10
more than 4 layers of granulosa cells although they occasionally processed small antral cavity
11
[10].The isolated PFs (SPFs and LPFs) were washed in the isolation and washing medium
12
containing minimum essential medium (MEM) supplemented with bovine serum albumin
13
(BSA, 0.3%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and
14
gentamicin (50µg ⁄ mL).
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A combined mechanical and enzymatic method developed in our laboratory was used
Only viable PFs as assessed by trypan blue staining method [11] were used for
16
culture. In brief, PFs were incubated in 0.04% trypan blue for 2 min at room temperature. PFs
17
stained with trypan blue were considered as dead (Fig. 3) and unstained PFs were considered
18
as alive (Fig. 4).The isolated PFs (two to three in a group) were transferred in 100µL droplets
19
of the culture medium under paraffin oil in a 35-mm petri dish and cultured in a CO2
20
incubator (38.5°C, 5% CO2 in air, 90–95% relative humidity) for 7 days (LPFs) or 14 days
21
(SPFs). The control culture medium was MEM supplemented with BSA (0.3%), glutamine (2
22
mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM), insulin–selenium–transferin (1%)
23
and gentamicin (50 µg ⁄ ml) and FSH-P (7µg ⁄ ml; biological potency = 7 U⁄ mg; F2293; LH
24
≤1%). The first day of culture was designated as Day-0. The medium was replenished at the
25
end of Day-2 and Day-4 for 7 day culture and additionally at the end of Day-6, Day-8, Day-
26
10 and Day-12 for a 14 day culture.
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Follicle diameters (µm) were assessed by measuring the distance between 2 sides of
28
the follicle, straight through the center of the oocyte using an eye piece micrometer fitted on
29
the stero zoom microscope (magnification X200) [12]. Oocyte diameters included zona-
30
pellucida thickness as it was reported that the formation of the zona pellucida always
31
occurred during the PF stage [13].The final diameter of the follicles was recorded together
32
with the presence and absence of an antral cavity (a visible translucent area within the
33
granulosa cell mass (Fig. 5). The growth rate measured was the cumulative growth rate.
3
ACCEPTED MANUSCRIPT The growth, survival rates of PFs and growth rate of oocytes in enclosed SPFs were
1 2
calculated as described earlier [9]:
3 4
1.
of follicle] ⁄ seven (LPFs) or fourteen (SPFs).
7 8 9 10
2.
Survival rate (%) = [No. of viable follicles at the end of culture) ⁄ no of viable follicles put into culture] ×100.
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Growth rate of follicle (µm⁄ day) = [Final diameter (µm) of follicle - Initial diameter (µm)
3. Growth rate of oocyte (µm ⁄ day) = [Final diameter (µm) of oocyte - Initial diameter (µm) of oocyte] ⁄ seven (LPFs) or fourteen (SPFs). Oocyte recovery and maturation
At the end of the culture, all of the healthy SPFs and LPFs were cautiously opened
12
mechanically using (26G) hypodermic needles attached to a 2mL syringe barrels under a
13
stereo zoom microscope. Oocytes with a homogeneous cytoplasm and surrounded by at least
14
two layer of granulosa cells were selected for in vitro maturation (IVM). IVM was performed
15
as described earlier [14]. In brief, oocytes were washed three times in washing medium
16
(TCM 199 + 10% FBS, 50µg/ml gentamicin) and then twice in the maturation medium
17
(TCM-199 + FBS (10%) + Follicle Stimulating Hormone-ovine (FSH-O, 10 µg/ml) + 50
18
µg/ml gentamicin). Oocytes (6-8) were matured in 50µL maturation medium droplets of
19
TCM-199 + FBS (10%) + Follicle Stimulating Hormone-ovine (FSH-O, 10 µg/ml) + 50
20
µg/ml gentamicin under oil in a 35-mm Petri dish in a CO2 incubator (38.5◦C, 5% CO2 in air,
21
90%–95% relative humidity) for 24 h. The mineral oil was pre-equilibrated with the culture
22
medium. Oocytes with an expanded cumulus cell mass (Fig 6) and with an extruded first
23
polar body in the perivitelline space (Fig 7) were considered as matured [15].
24
Levels of ROS
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Levels of ROS were determined for the culture media by the spectrofluorimetric
26
method, using the 20, 70-dichlorofluorescein diacetate (DCF-D) assay [16]. The medium was
27
incubated with 5mL of DCF-D (1 mM), and the oxidation of DCF-D to fluorescente
28
dichlorofluorescein was measured for ROS detection. Dichlorofluorescein fluorescence
29
intensity emission was recorded at 520nm (with 480 nm excitation) 120 minutes after the
30
addition of DCHFDA to the medium.
31
Hormone assay
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The spent medium obtained at the end of Day-7 and Day-14 for LPFs and SPFs
33
respectively were pooled per plate and stored at -80 ºC for hormonal assays. E2 and P4
34
concentrations were determined by ELIAS kits (commercial kits for clinical use in humans, 4
ACCEPTED MANUSCRIPT 1
Diagnostics Biochemical’s Pvt Inc., Ontario, Canada). The calibrator ranges were 20 to 3200
2
pg/mL and 0.15 to 20 ng/mL for E2 and P4 respectively. Twenty five microlitres of samples
3
were used in quadruplicate. All samples were run in one assay to avoid inter-assay variation
4
and intra-assay variations were 2.4 and 2.7% for E2 and P4 respectively.
5
Assessment of SOD activity Twenty PFs from each treatment group were collected and used on the same day for
7
SOD activity assay. Enzymatic extracts of in vitro cultured SPFs and LPFs at the end of final
8
culture period (i.e 7 days for SPFs and 14 for LPFs) were obtained by homogenizing then in
9
cold 20 mM HEPES buffer followed by centrifugation for 5 min at 4 °C. Total SOD activity
10
(inhibition rate %) was determined by the SOD Assay Kit-WST (Sigma-aldrich, USA)
11
according to the manufacturer’s recommendations. The assay was replicated three times. The
12
absorbance read at 450nm with a micro plate reader.
13
Selection of levels of metabolic stressors
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The types and concentrations of metabolic stressors used in this study were based on
15
earlier in vivo and in vitro studies [5-8, 14, 15] in our laboratory. In brief, the mean follicular
16
fluid ammonia, urea, NEFA and BHB levels were 132 Vs 157µM; 4.0 Vs 6.0 mM; 78.0 Vs
17
99.0µM; and 0.5 Vs 0.72mM in post parturient and high protein diet scenario (metabolically
18
stressed ewes) respectively. The ammonia level ranged from 94 to 412 µM, urea from 3 to
19
8.6mM, NEFA from 68 to 256 µM and BHB from 0.42 to 0.94 mM in metabolic stressed
20
ewes. We also reported that the mean basal NEFA level in ewe follicular fluid was 70.4 µM,
21
and the oleic, palmitic and stearic acids were the three predominant free fatty acids in
22
follicular fluid and the average relative presences of these NEFAs were 40%, 25% and 15%
23
respectively. We used the following composition of NEFA in the present study: a) Basal
24
NEFA (70µM): stearic acid, SA (10µM)+Palmitic acid, PA(20µM)+oleic acid, OA(40 µM),
25
b) Medium combo (140 µM): SA (20µM)+ PA(40µM)+ OA(80 µM), c) High combo (210
26
µM): SA (30µM)+PA(60µM)+OA(120µM), d) Very high Combo (280µM): SA(40
27
µM)+PA(80µM)+OA(160µM).
28
Experiments
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Four different experiments, to investigate the influence of inclusion of different
30
concentration of ammonia [0 (control), 100 (Basal level), 150, 200, 250, 300 and 400µM],
31
urea [0 (control), 4 (basal level), 4.5, 5, 5.5,6, 7 and 8mM], NEFA [0 (control), Basal NEFA,
32
High-Combo and Very high-combo] and BHB [0(control), 0.5 (basal), 0.75, and 1µM] on the 5
ACCEPTED MANUSCRIPT 1
in vitro development of ovine PFs were conducted. Each of the experiments was replicated
2
for 8 times each for SPFs and LPFs. Each replicate consisted of twelve 100µL droplets with
3
2-3 PFs for each treatment.
4 5 6
Statistical analysis
7
by a prospective, randomized study. The interactions between follicle diameter and treatment
8
as well as replicate and treatment were performed as per the model described earlier [12]
9
however as we did not get any significant interaction, we omitted from the final analysis.
10
Significant differences in experiments between the growth rates of PFs and oocytes, survival
11
rates of PFs, maturation rate of oocytes, hormone production and SOD activity were analyzed
12
by ANOVA and the respective means were compared using Tukey’s test where treatment
13
was entered as a fixed factor, and replicate as a random factor. The differences between the
14
mean values of all parameters were tested by t-test wherever the comparison was made
15
between SPFs and LPFs. The percentage values were transformed by arcsine square root
16
before analysis. Difference was considered to be significant at p< 0.05. The computer assisted
17
statistical software package (Graph Pad Prism, San Diego, CA, USA) was used for analyzing
18
the data.
19
Results
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The effects of each factor on the follicular features (growth ⁄ survival) were evaluated
The effects of ammonia, urea, NEFA and BHB on in vitro development of SPFs and
21
LPFs are presented in Tables 1-4. In all the four experiments LPFs exhibited better frequency
22
of survival, rates of growth and maturation of oocytes to MII stage as well as estrogen
23
production (Tables 1-4). The frequency of survival, rate of growth of PFs' and maturation of
24
oocytes to MII stage began to decrease significantly on exposure to 300 µM ammonia and
25
was further decreased at 400µM in both SPFs’ and LPFs’(Table 1). While the estrogen
26
production was reduced at 400µM of ammonia, SOD activity started to decline significantly
27
at 200µM concentration (Table1).However, the progesterone production was not adversely
28
affected by exposure to ammonia (Table 1).
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The frequency of survival, rate of growth of PFs' and maturation of oocytes to MII,
30
estrogen production and SOD activity began to decrease significantly on exposure to 8mM of
31
urea in both SPFs’ and LPFs’(Table 2). However, the progesterone production was not
32
adversely affected by exposure to urea (Table 2).
6
ACCEPTED MANUSCRIPT The frequency of survival, rate of growth of PFs' and maturation of oocytes to MII
2
stage began to decrease significantly on exposure to High combo NEFA and was further
3
decreased at Very high combo NEFA in both SPFs’ and LPFs’ (Table 3), while the estrogen
4
production was reduced at Medium combo NEFA compared with Basal NEFA (Table 3).
5
Further, significant reduction in estrogen production was observed when LPFs were treated
6
with high level of NEFA compared to medium level NEFA, SOD activity started to decline
7
significantly at Medium combo NEFA concentration and was maximum at very high combo
8
NEFA in both SPFs’ and LPFs’ (Table 3). However, the progesterone production was not
9
adversely affected by exposure to ammonia (Table 3).
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The rate of growth of LPFs and survival rate of both SPFs and LPFs were
11
significantly decreased on exposure to 0.75µM BHB compared to lower levels (Table 4).
12
Further increment of BHB level to 1µM level decreased the survival rate of only LPFs. The
13
rate of growth and maturation of oocytes to MII stage began to decrease significantly on
14
exposure to 1.0µM BHB concentration (Table 4), while SOD activity started to decline
15
significantly at 0.75µM concentration and was maximum at 1.00µM in both SPFs’ and LPFs’
16
(Table4). However, the estrogen and progesterone production was not adversely affected by
17
exposure to BHB (Table 4).
18
Discussion
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The impact of metabolic stress on the preantral phase of folliculogenesis in domestic
20
animals is unknown. Therefore, we studied the effect of metabolic stressors on PFs
21
(representing 90% of ovarian follicle population) using ovine follicles cultured in vitro.
22
During the journey of PFs to ovulation, follicles might be under different stresses (heat stress,
23
nutritional stress, disease or other factors) that could impair the developmental competence of
24
follicle-oocyte complexes [17]. Similarly, primary follicles exposed to stress were less
25
competent to produce ample quantities of estrogens and progesterone and such follicles
26
contained poor quality oocytes [18].
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It was reported that exposure of bovine oocytes to ammonia concentrations of 29 to
28
356mM during IVM did not adversely influence oocyte development which indicated that
29
bovine oocytes could tolerate elevated concentrations of ammonia during IVM as determined
30
by the subsequent embryonic development in vitro [19]. In contrast, we observed that
31
maturation rates of oocytes retrieved from cultured PFs’ in the present study as well as those
32
retrieved from antral follicles in an earlier study [16] were significantly lowered when
33
exposed to 250µM ammonia. Similarly, it was reported that the high urea level of serum
7
ACCEPTED MANUSCRIPT 1
resulted in penetration of urea into follicles; this did not impair the development of such
2
follicles [20]. However, our results suggested that PF growth was impaired when exposed to
3
8 mM level of urea. Long-term exposure of elevated NEFA concentration (720µM) to murine follicles
5
was reported to influence the follicular growth, with the most marked effect induced by the
6
high stearic acid (280µM) treatment [12]. The same authors also reported that oocytes
7
originated from the NEFA-exposed follicles showed notably low oocyte developmental
8
competence. High NEFA also reported to cause amended glucose metabolism and increased
9
beta-oxidation, and its outcome was higher concentration of ROS production [21]. Similarly
10
exposure of bovine oocyte to high concentration of NEFA (425µM) was reported to affect β-
11
oxidation or lipogenesis and vital mechanisms for developmental competence of oocytes
12
[22]. It was reported that oocytes matured under high saturated NEFA environment showed
13
significantly distorted metabolic pathways at both gene transcription and gene function levels
14
[12]. We observed significant changes in terms of growth and survival of PFs when cultured
15
in medium containing high combo NEFA. In mice, elevated NEFA concentration negatively
16
affected pathways involved in apoptosis, lipid metabolism, oxidative stress and
17
steroidogenesis, which was confirmed by P4, oestradiol and inhibin B analyses in spent
18
medium [12]. Valckx and her co-workers [23] cultured murine early secondary PFs up to Day
19
13, followed by oocyte isolation, fertilisation and embryo culture. When these follicles are
20
exposed to elevated NEFA concentrations (720µM) throughout follicle growth or only during
21
the final maturation phase, long-term exposure (13 days) severely impaired oocyte
22
developmental competence when compared with short-term NEFA exposure. This
23
strengthened our observation that not only final oocyte maturation, but also the preceding
24
period of follicular and oocyte growth were influenced by metabolic stressors. It should also
25
be noted that simple stomached animals could have lower tolerance for some of these
26
metabolites; ruminants might be less sensitive since these metabolites were normally
27
produced in significant quantities during digestion in rumen and passed into circulation.
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Ketone bodies like BHB etc were negatively associated with fertility (disturbances in
29
LH pulse frequency, low growth of the follicle, and decreased progesterone and estradiol
30
secretions) [24]. Our findings were in accordance to the earlier report [25] wherein it was
31
observed that the very high BHB concentrations as well as the concomitant hypoglycaemic
32
conditions seemed to be responsible for adverse effect on development competence of
33
oocyte. It was also reported that in bovine more than 50mg/l concentration of BHB in
34
follicular fluid had been negatively correlated with the percentage of good-quality oocytes 8
ACCEPTED MANUSCRIPT 1
and their developmental potential [26]. This was in agreement with our findings wherein the
2
SPFs and LPFs exposed to high BHB concentration showed less development and survival
3
rates. In an earlier study it was reported that there was a reduction in progesterone
5
production and no change of estrogen production under high NEFA in early PFs was
6
recorded [12]. However we observed no change in progesterone and reduction in estrogen
7
production under metabolic stress. Less estrogen production in the present study might be
8
attributed to low aromatase activity due to metabolic stressors as examined by ELISA (Nandi
9
S, unpublished data) or by low expression of caveolin 1, a regulator of aromatase activity [27].
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The detrimental effects of the stress on PFs might also be due to generation of high
12
concentration of ROS that affected the growth of PFs as well as oocytes [16]. Moreover, the
13
metabolic stress might led to higher expression or upregulation of proapoptotic genes and
14
cellular apoptosis as reported earlier [28, 29]. The increased concentration of metabolic
15
stressors in all the tested treatments in the present study caused a decline in follicle viability
16
and growth, signifying that this incident might possibly arbitrated by ROS.
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Our results differed from a possibility as suggested earlier that oocytes within PFs
18
were completely insensitive to the metabolic stressors [30]. The same authors reported that
19
the oocytes were exposed to a short period of time in in-vitro system whereas in in-vivo the
20
oocytes were exposed to these concentrations for several days or even weeks. According to
21
the same authors in the ideal model, PFs should be cultured in metabolic stressors for several
22
weeks.
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Subsequent build-up of ammonium, generated from the breakdown of amino acids
102
and glutamine in the culture medium, impaired cell growth [31, 32]. Ammonia concentrations
103
in fresh IVM control medium were below detectable levels and increased from 4 to 7 mM at
104
the end of the 2 days culture period (the media were replenished every 2 days) in the present
105
study. We had used medium devoid of glutamine and used sodium pyruvate as energy source.
106
This might be the cause of lower amount of ammonia generated in the IVM medium in the
107
present study. We used periodic replacement of medium as the gradual increase in the culture
108
medium volume through the addition of fresh medium would provide new nutrients and also
109
maintain the medium with substances produced in the different compartments of the follicle
110
[33]. Periodic addition of medium (small supplementation method) was recommended
111
because it was more practical, maintained survival and promoted the development of
112
preantral follicles in vitro [34].
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ACCEPTED MANUSCRIPT The present study had shown, for the first time, ovarian follicles even at the smaller
2
size were affected by changing milieu of metabolites. All the metabolic stressors (ammonia,
3
urea, NEFA and BHB) impaired the functions of ovarian PFs and their enclosed oocytes at
4
the physiological levels observed during metabolic stress conditions. Ammonia, urea, NEFA
5
and BHB caused inhibition of survival and growth of in vitro cultured ovine PFs and
6
enclosed oocytes at the levels of 300 µM, 8mM, high combo level of NEFA and 0.75 µM
7
respectively The results of the present study might contribute to the identification of the
8
mechanisms and factors involved in reduced fertility due to metabolic and nutritional stress in
9
ruminants. Acknowledgement
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We are grateful to the Director, NIANP, Bangalore for providing the necessary facilities. We thank Mr. Gyan Prakash for technical assistance. Financial help from the
15
Department
16
BT/PR7131/AAQ/1/526/2012) is gratefully acknowledged. We declare no conflicts of
17
interests.
of
Biotechnology,
18
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India
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methods. Animal Reproduction Science 2015,161: 23–31.
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ACCEPTED MANUSCRIPT
ROS (fluorescence units) Estrogen (pg/mL) production Progesterone (ng/mL) production
77.5±3.5a
LPF SPF* LPF* SPF LPF SPF* LPF* SPF
81.2±3.9a 2.9±0.21a 11.6±0.34a 3.7±0.2 a 3.4±0.4 a 46.5±2.7a 62.6±3.6a 15.3 ±3.4 a
77.3±3.6a 2.8±0.21a 11.4±0.32a 3.6±0.3 a 3.4±0.5 a 44.3±1.8a 61.8±1.6a 15.8 ±3.1 a
77.3±3.3a 2.8±0.19a 9.8±0.16a 3.3±0.4 a 3.1±0.1 a 45.6±1.7a 60.6±3.2a 15.8 ±3.4 a
LPF SPF*
16.5 ±2.4 a 98.6±7.8a
15.9 ±3.0 a 94.3±6.0a
16.1 ±2.5 a 92.9±5.8a
15.8 ±2.7 a 93.1±5.4a
21.4 ±3.1 b 92.3±4.6a
22.1 ±2.2b 92.6±3.3a
26.7 ±2.9c 68.6±2.8b
LPF* SPF
148.4±9.8a 3.4±0.7a
148.0±8.2a 3.4±0.9a
149.8±7.6a 3.2±0.3a
128.4±4.5a 3.2±0.1a
128.7±6.2a 3.2±0.5a
120.1±4.3a 3.2±0.3 a
87.8±5.8b 3.2±0.7 a
LPF SPF
4.6±0.1a 56.2±1.2a
4.6±0.1a 55.4±0.9a
4.5±0.2a 49.3±1.5a
4.3±0.3a 38.4±2.3b
4.3±0.2a 36.1±1.6b
4.2±0.3a 20.4±0.3c
4.2±0.2a 18.1±0.8c
40.1±4.2b
38.4±3.8b
22.7±1.4c
16.6±1.7c
73.3±3.1a
200 (n=208)
250 (n=204)
300 (n=216)
400 (n=216)
74.5±2.5a
70.4±3.1a
62.7±2.4b
47.1±2.3c
76.5±2.3a 2.6±0.20a 9.1±0.23a 3.3±0.2 a 3.2±0.3 a 42.4±3.6a 58.2±2.0a 15.7 ±3.2 a
72.0±3.2a 2.6±0.22a 8.8±0.25a 2.8±0.3 a 2.9±0.4 a 30.3±1.9b 47.1±1.7b 20.3 ±2.5b
66.4±2.7b 2.1±0.17b 3.9±0.16b 1.3±0.3 b 1.4±0.6 b 28.6±0.8b 42.4±0.7b 21.6 ±3.2b
45.3±2.7c 1.3±0.12c 2.4±1.7c 0.7±0.2 c 0.9±0.3 b 15.7±1.5c 21.7±0.4c 25.4 ±2.5c
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150 (n=224)
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Growth (in enclosed follicles) rate of oocyte (µM/day) Maturation rate of Oocytes (%)
SPF
100-Basal value (n=192) 75.2±3.2a
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Growth rate of PFs (µM/day)
0-Control (n=192)
EP
Survival rate of PFs, %
Concentration of ammonia, µM
AC C
Parameter studied
Follicle type
RI PT
Table 1: Effect of ammonia on small and large PFs cultured in vitro
SOD enzyme activity (Inhibition rate %) LPF 61.2±3.1a 60.1±2.5a 56.4±1.8a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
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Maturation rate of Oocytes (%) ROS (fluorescence units)
Estrogen (pg/mL) production
74.6±2.2a 77.3±2.9a 2.7±0.23a 10.5±0.21a 3.3±0.3a 3.7±0.9 a 42.8±2.7a
72.1±3.2a 74.2±3.1a 2.6±0.20a 9.4±0.20a 3.3±0.4a 3.4±0.3 a 40.5±2.5a
71.3±2.1a 74.3±2.6a 2.7±0.16a 9.1±0.19a 3.1±0.3a 3.4±0.2 a 40.2±3.2a
70.3±2.1a 74.4±2.4 a 2.7±0.10a 9.3±1.4a 3.0±0.3 a 3.2±0.4 a 38.6±3.4a
68.2±1.8a 70.2±2.7a 2.6±0.27a 9.03±1.5a 3.0±0.5a 3.1±0.1a 36.6±2.4a
54.3±2.4 b 52.3±1.4 b 2.1±0.21b 7.04±1.1b 1.9±0.8b 1.7±0.4b 20.5±3.2b
62.6±2.4a
60.3±3.8a
60.2±2.2a
60.4±2.1a
58.5±3.4a
37.1±2.3b
75.3±3.1a 77.2±3.5a 2.9±0.22a 10.9±0.31a 3.6±0.2a 4.1±0.6 a 44.8±2.6a
LPF*
66.4±2.4a
63.6±3.2a
SPF LPF
12.5 ±4.4 a 14.1 ±2.4 a
13.1 ±2.1 a 14.8 ±2.0 a
13.4 ±2.4 a 15.5 ±3.5 a
13.7 ±1.2 a 15.2 ±1.7 a
14.3 ±3.5a 16.4 ±3.2 a
14.8 ±2.2a 16.8 ±2.5a
22.4 ±4.5b 28.7 ±3.0b
26.1±2.3c 30.5±2.1c
SPF*
99.3±6.3a
98.7±5.2a
97.5±8.2a
98.3±5.9a
97.4±6.3a
97.1±6.6a
96.6±7.9a
75.3±4.8b
138.2±8. a 3.4±0.4 a 4.5±0.1a 52.6±2.8a 55.6±1.9a
135.7±5.3a 3.2±0.3 a 4.5±0.2a 51.2±3.7a 54.1±2.3a
132.9±6.9a 3.2±0.4 a 4.5±0.2a 51.8±3.2a 54.7±2.8a
133.2±6.1a 3.0±0.3a 4.4±0.2a 50.7±3.1a 52.5±2.2a
93.1±7.9b 3.1±0.2a 4.3±0.2a 43.8±2.5b 44.7±2.9b
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Growth (in enclosed follicles) rate of oocyte (µM/day)
8 (n=216)
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Growth rate of PFs (µM/day)
7 (n=204)
SPF LPF SPF* LPF* SPF LPF SPF*
EP
Survival rate of PFs, %
4.5 (n=204)
4-Basal value (n=204) 74.6±1.7a 76.3±3.0a 2.9±0.20a 10.7±0.34a 3.6±0.4a 4.2±0.7 a 43.4±2.9a
Parameter studied
0-Control (n=192)
Concentration of urea (mM) 5 5.5 6 (n=216) (n=216) (n=224)
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Follicle type
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Table 2: Effect of urea on small and large PFs cultured in vitro
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LPF* 147.8±9.5a 147.3±8.6a 146.2±7.5a a a SPF 3.4±0.7 3.4±0.2 3.5±0.8 a Progesterone (ng/mL) production LPF 4.6±0.2a 4.6±0.3a 4.5±0.1a a a SPF 55.4±3.6 56.2±2.4 53.4±4.2a SOD enzyme activity (Inhibition rate %) LPF 60.7±1.9a 60.4±2.5a 56.1±1.6a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
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Table 3: Effect of NEFA on small and large PFs cultured in vitro
Parameter studied
Concentration of NEFA (µM) 0-Control (n=204)
Basal NEFA (n=204)
SPF 79.4±2.5a 73.5±2.2a a LPF 84.3±2.4 77.1±2.6a SPF* 2.9±0.28a 2.8±0.25a Growth rate (µM/day) a LPF* 10.9±0.29 10.7±0.31a 3.5±0.3a SPF 3.6±0.2a Growth (in enclosed follicles) rate of oocyte (µM/day) LPF 4.3±0.2a 4.2±0.3a a SPF* 41.3±1.9 42.5±2.5a Maturation rate of Oocytes (%) LPF* 63.5±2.3a 62.4±2.7a a SPF 8.2 ±3.4 8.5 ±23.1 a ROS (fluorescence units) LPF 11.4 ±2.1 a 11.9 ±1.0 a a 94.2±5.2a SPF* 94.6±3.2 Estrogen (pg/mL) production LPF* 140.0±8.6a 143.7±7.2a SPF 3.7±0.4a 3.6±1.2a Progesterone (ng/mL) a production LPF 4.4±0.1 4.4±0.8a SPF 59.3±2.2a 55.4±1.5a SOD enzyme activity a (Inhibition rate %) LPF 57.4±4.5 57.6±2.3a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
Medium Combo (n=208)
High Combo (n=216)
Very high Combo (n=224)
74.1±1.1a 79.4±1.4a 2.7±0.18a 9.1±0.9a 3.4±0.5a 3.9±0.3a 40.4±2.4a 60.6±3.6a 9.1 ±4.4 a 12.4 ±2.5 a 80.6±2.9b 117.7±9.3b 3.5±0.6a 4.3±0.3a 31.4±1.3b 37.6±1.6b
63.3±2.1b 69.6±2.9b 2.4±0.16b 4.4±0.24b 2.1±0.9b 2.4±0.4b 27.4±2.4b 40.7±2.6b 16.8 ±5.2 b 20.4 ±4.7 b 70.1±1.6b 80.3±6.2c 3.3±0.1a 4.3±0.5a 20.3±1.2c 22.4±2.2c
53.4±2.2c 54.4±2.7c 2.1±0.20c 4.3±0.22c 0.4±0.6c 0.9 ±0.2c 13.3±1.2c 18.5±1.4c 18.3 ±2.5b 22.4 ±2.2 b 68.8±2.6b 77.7±4.5c 3.2±0.1a 4.2±0.3a 18.7±1.3c 21.5±2.3c
AC C
EP
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M AN U
SC
Survival rate of PFs, %
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Follicle type
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Table 4: Effect of BHB on small and large PFs cultured in vitro
Parameter studied
1.00 (n=222) 66.1±1.7b 69.6±0.9c 2.2±0.1a 3.2±1.6b 2.6±0.4 b 3.7±0.2 b 28.6±1.4 b 40.4±2.5b 16.7 ±2.2 b 20.2 ±2.5 b 95.1±5.1a 132.6±8.6a 3.6±0.09a 4.0±0.1 a 36. ±2.4c 20.1±3.4c
AC C
EP
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M AN U
SC
0-Control (n=208) SPF 80.1±2.4a Survival rate of PFs, % LPF 83.3±1.4a SPF* 2.8±0.4a Growth rate (µM/day) LPF* 10.6±2.1a SPF 3.5±0.4 a Growth (in enclosed follicles) rate of oocyte (µM/day LPF 4.0±0.6 a SPF* 43.6±2.7a Maturation rate of Oocytes (%) LPF* 62.6±2.6a SPF 10.8 ±1.9 a ROS (fluorescence units) LPF 12.1 ±1.4 a SPF* 96.2±4.2a Estrogen (pg/mL) production LPF* 142.3±9.4a SPF 3.9±0.1a Progesterone (ng/mL) production LPF 4.2±0.1a SPF 60.2±2.5a SOD enzyme activity (Inhibition rate %) LPF 63.1±3.6a SPF and LPF refer to small and large preantral follicles, respectively. Values are Mean ±SEM. Values with different superscripts in the same row differ significantly (P < 0.05). *Denotes the significance between SPF and LPF
Concentration of BHB (µM) 0.5-Basal value 0.75 (n=216) (n=218) 82.4±3.7a 70.6±3.6 b 81..1±0.6a 75.4±1.4b 2.7±0.6a 2.4±0.3a a 9.9±1.7 6.4±1.4b 3.4±0.6 a 3.2±0.5 a 4.1±0.3 a 4.3±0.4 a a 41.6±2.7 40.4±2.5a 62.7±2.5a 58.2±3.4a a 11.4 ±2.8 15.4 ±4.4 b 12.8 ±2.5 a 18.7 ±3.9 b a 95.7±2.5 94.4±3.8a 139.3±8.4a 135.5±7.5a a 3.7±0.2 3.8±0.1a 4.0±0.2a 4.1±0.1a a 57.4±1.6 40.7±1.9b 59.3±2.9a 34.2±2.4b
RI PT
Follicle type
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ACCEPTED MANUSCRIPT
Fig. 1. Small Preantral follicle
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Fig. 2. Large preantral follicle
Fig. 4. Preantral follicle (alive)
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Fig. 3. Preantral follicles (Dead)
Fig. 5. Follicle with antrum
Fig 6. Oocytes with expanded cumulus cell mass
Fig 7: Oocyte with extruded first polar body
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ACCEPTED MANUSCRIPT
Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes S. Nandi*, S.K.Tripathi, P.S.P.Gupta, S. Mondal
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ICAR-National Institute of Animal Nutrition and Physiology, Bangalore-560030, India
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*Corresponding author: ICAR-National Institute of Animal Nutrition and Physiology (ICARNIANP), Bangalore 560030, India; Email: [email protected]
Highlights
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Ovarian follicles even at the smaller size were affected by changing milieu of metabolites in ovarian tissues and follicular fluid. Increased levels of ammonia, urea, NEFA and BHB impaired the functions of ovarian preantral follicles and their enclosed oocytes
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