- Email: [email protected]
Vascularization to preovulatory follicle and corpus luteum-a valuable predictor of fertility in dairy cows
Accepted Manuscript Vascularization to preovulatory follicle and corpus luteum-a valuable predictor of fertility in dairy cows Emy E. Varughese, Parkash S. Brar, Sarvpreet S. Ghuman PII:
S0093-691X(17)30373-4
DOI:
10.1016/j.theriogenology.2017.07.042
Reference:
THE 14208
To appear in:
Theriogenology
Received Date: 9 May 2017 Revised Date:
21 July 2017
Accepted Date: 27 July 2017
Please cite this article as: Varughese EE, Brar PS, Ghuman SS, Vascularization to preovulatory follicle and corpus luteum-a valuable predictor of fertility in dairy cows, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.07.042. 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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Vascularization to preovulatory follicle and corpus luteum- a valuable predictor of fertility
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in dairy cows
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Emy E. Varughese*, Parkash S. Brar, Sarvpreet S. Ghuman
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Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, India
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* Corresponding author: Tel: +1 614-592-5900
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Email: [email protected]
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Present address: 5300 Riverside Dr, Upper Arlington, Ohio, USA 43220
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Abstract
The aim of the present study was to predict pregnancy rate based on vascularization to follicle
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and corpus luteum (CL). 26 Holstein Friesian cows were synchronized using Ovsynch protocol.
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On day 10 of the protocol, vascularization and morphological characteristics [sectional area
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(SA), volume (V) and mean diameter] of follicle was assessed and animals underwent artificial
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insemination (AI). Morphological evaluation and vascularization to CL was assessed on day 12
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and 21 following AI and blood samples were obtained for estimation of plasma progesterone
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(P4). Pregnancy diagnosis was performed on day 60 of AI and was classified as normal,
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complicated and non-pregnant. The overall conception rate was 76.92 % (20/26); normal
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pregnancy was 53.85% (14/26). Complications observed in pregnancy were intrauterine growth
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retardation, late embryonic death and infection. Cows with a highly vascularized follicle (> 550
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pixel2) underwent a normal pregnancy, whereas those that had moderately (250 to 550 pixel2)
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and poorly (< 250 pixel2) vascularized follicle experienced complicated pregnancy or remained
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non-pregnant, respectively. On day 12, there was no significant variation (P > 0.05) between
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mean diameter, SA, V, luteal blood flow (LBF) or plasma P4 concentration among CL of cows
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that remained pregnant (PCL), non-pregnant (NPCL) or that had a complicated pregnancy
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(CPCL). LBF alone was not beneficial in differentiating among the three groups (P > 0.05), but
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assessment of LBF along with turbulence to blood flow in day 21 CL proved highly valuable due
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to an increased turbulence in CPCL (66.67%) compared to PCL (16.67%). Assessment of
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turbulence and LBF on day 12 and 21 can also be used to predict luteolysis with accuracy.
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Keywords: Doppler ultrasonography; Follicle; Corpus luteum; Blood flow; Pregnancy.
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1. Introduction
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A calf per animal per year is the most favorable realization of every farmer/dairy enterprise. A
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successful pregnancy involves several important stages. Firstly, an ovarian follicle must develop
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and ovulate an oocyte capable of being fertilized and undergo embryonic development.
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Secondly, the oviductal and uterine environments must be suitable for gamete transport,
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fertilization, and subsequent embryonic development. Finally, the corpus luteum (CL) must
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function for a sufficient period of time for maternal recognition of pregnancy and maintain
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gestation [1]. Hemodynamic changes are involved in the cyclic remodeling of ovarian tissue that
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occurs during final follicular growth, ovulation and new CL development [2] during which
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angiogenesis plays a crucial role in the maintenance of ovarian structures.
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Transrectal Doppler ultrasonography has been utilized increasingly for research and clinical
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studies of ovarian hemodynamics involving follicle and CL in large farm animals [3]. Blood
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flow determinations of individual preovulatory follicles provide an important index on the
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intrafollicular environment and may predict the developmental competence of the corresponding
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oocyte [4,5]. A study on 39 Holstein heifers proved that blood flow at the time of artificial
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insemination (AI) was greater in the preovulatory follicle wall of heifers that became pregnant
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than in heifers that did not [6]. Relationships between the echogenicity of CL and progesterone
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(P4) concentrations have already been made with conventional gray-scale ultrasound in heifers
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[7] and in mares [8] and an increased blood flow has been proposed to augment the transfer of
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the embryonic signal for CL maintenance and for maternal recognition through the utero-ovarian
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pathway [9].
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Therefore, assessment of blood flow to ovarian structures (follicle and CL) has been shown to
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give preliminary information regarding pregnancy rate [6]. However continuous assessment of
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follicle, CL and sustenance of pregnancy has not been studied. Early prediction of nature of
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pregnancy based on blood flow to follicle and CL will prove to be a boon for the dairy industry.
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Infertility and complications in pregnancy remains to contribute significantly to reduced
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conception rates in dairy farms. Since follicle and CL are ovarian structures easily visualized by
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ultrasonography and indispensable for a successful pregnancy, our study aimed at assessing
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blood flow to follicle and CL to predict fertility in dairy cows.
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2. Materials and methods
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2.1 Animals
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The study was conducted on twenty-six healthy, reproductively normal and regular cycling
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postpartum Holstein Friesian cows (body weight: 300 to 350 kg, body condition score (BCS): 3.5
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to 4, parity: 2 to 3). The cows were housed in an organized dairy farm in semi-loose housing
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system and were reared on green fodder (30 to 40 kg), wheat straw (2 kg), concentrate feed,
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mineral mixture and ad libitum drinking water.
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2.2 Ovulation synchronization (Ovsynch)
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Irrespective of the stage of estrus cycle, the cows were subjected to an ovulation synchronization
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protocol. Cows were treated with a GnRH analogue (Buserelin acetate, ReceptalTM, Intervet,
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India, im) on day 0 (20 µg) and day 9 (10 µg) along with a synthetic prostaglandin F2α analogue
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(Cloprostenol Sodium, VetmateTM, Vetcare, Bangluru, India, 500 µg im) on day 7. 3
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2.3 Study design
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On the day of estrus (Day 10, of Ovsynch protocol), transrectal examination and ultrasonography
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were used to locate and measure the size of follicle respectively. Colour flow mode (CFM) was
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used to assess the blood supply to the follicle, and timed AI was performed. Blood flow of the
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follicle was reassessed on day 11 (24hrs following AI), and AI was repeated if the follicle had
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not ovulated. Size and blood flow of CL was assessed on day 12 and 21 following AI
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(represented as day 12 and 21 respectively). Blood samples were collected on day 12 and 21 for
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estimation of P4. Pregnancy diagnosis was performed in all the animals on day 60 of AI using
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ultrasonography.
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2.4 Brightness mode (B-mode) and Doppler ultrasonography
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Transrectal ultrasonography was carried out using a battery operated B-mode ultrasound scanner
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equipped with a 7.5 MHz, linear-array transducer (Exago, ECM - Noveko International
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Inc., Angoulème, France) for assessing the size of follicle/CL. The frozen optimal scan images
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were used to determine the diameter (average of maximum length and transverse diameter) with
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the help of built-in, on-screen callipers. The volume (V) of the follicle and CL were estimated
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using the following equation for a modified prolate ellipsoid: V = 0.523 × A × B2, in which A
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represents the maximum length and B represents the transverse diameter [10]. CL were described
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as CL with a single (central or eccentric)/multiple cavities or CL with incomplete luteinization of
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the follicular wall [11]. If a CL contained a cavity, volume of the cavity was determined and
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subtracted from the calculated CL volume except in CL with incomplete luteinization. In
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addition, the image obtained in a vertical plane from the apex to the base of the follicle/CL,
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designated as the overall image was used to determine the sectional area (SA) of the follicle/CL:
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SA= π/4 × (SD)2, where SD is the sectional diameter [10].
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Thereafter, color flow mode (4000 Hz pulse repetition frequency and 27.5 dB gain) of the
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scanner was used for blood flow mapping of follicle/CL. The red color indicated blood-flow
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toward the transducer’s face, and blue color indicated blood-flow away from the transducer’s
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face [12]. When the transducer was placed close to the follicle/CL, the blood flow captured by
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the transducer appeared as different color intensities on the monitor. As the velocity of the flow
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increased, the color intensity increased. Forward flow with turbulence resulted in yellow and
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backward flow with turbulence resulted in cyan (blue-green) [13]. Transducer was positioned at
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the maximal diameter of the follicle/CL to achieve the maximal number of color pixels in the
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recorded image. The blood vessels supplying the structures were evaluated based on their
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diameter into small (< 3mm), medium (3-6mm) and large (> 6mm).
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2.5 Quantification of blood flow
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Colored spots or pixel aggregates were selected from the images, extracted, and saved using
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Adobe Photoshop software (Adobe ® Photoshop ® CS3 Extended, Version 10.0, USA). Image J
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(Image J 1.45s, USA) was used to calculate total number of colored pixels, expressed as pixel2.
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2.6 Pregnancy diagnosis
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Pregnancy was confirmed at Day 60. Fetus appeared as an echogenic structure inside a non-
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echogenic structure [14] using B-mode ultrasonography. Once the fetus was detected, image was
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frozen and crown-rump length (CRL, a straight line between the fetal crown and the origin of the
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tail), trunk diameter (TD, at level of umbilical cord attachment) and skull diameter (SkD, the
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widest diameter) were measured and the image was recorded in a few normal and other cases.
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Blood flow velocity wave forms of the middle uterine artery were recorded in those cases which
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were suspected for fetal growth retardation/abnormalities and in normal cases for comparison.
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The middle uterine artery was located using CFM and switched to Pulsed-wave mode (4000Hz
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pulse repetition frequency, 12 dB gain, 50% power and 20 to 60 degrees Doppler angle) for
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determination of waveform. The waveform was evaluated for Resistance Index (RI) and
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Pulsatility Index (PI). A higher resistance or RI indicated lower perfusion to the particular organ
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and a high PI indicated decreased perfusion to distal tissues [12].
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Based on the blood supply of follicle/CL, fetal characteristics and blood flow velocity
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waveforms, pregnancy was classified as normal, complicated and non-pregnant.
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2.7 Blood sampling
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Jugular vein blood samples (10 ml) were collected in heparinized vaccutainer vials prior to
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ultrasonography on day 12 and 21. Plasma was separated immediately after blood collection by
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centrifugation at 3000 X g for 15 min. The plasma aliquots were stored at −20°C for estimation
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of P4.
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2.8 Estimation of P4
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Plasma P4 was assayed with a solid-phase radioimmunoassay using antisera raised in our
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laboratory [15]. Sensitivity of the assay was 0.1 ng/ml; intra- and inter-assay variation
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coefficients were 6.2% and 9.5%, respectively.
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2.9 Statistical Analysis
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Numerical data is expressed as Mean ± SEM. Statistical analysis was performed using SPSS 16
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[16]. Pearson’s correlation coefficient (r) tested the correlation between variables. One-way
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ANOVA was used to compare means within variables. Post hoc test (Tukey HSD) was used to
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3. Results
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Pregnancy diagnosis on day 60 revealed an overall conception rate of 76.92 % (20/26).
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3.1 Characteristics of blood flow of follicle and CL and its correlation with plasma concentration
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of P4 in:
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3.1.1 Pregnant cows (n=12).
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Follicular blood flow (FBF) on the day of AI was 841.33 ± 155.22 pixel2 (n=12). However, three
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cows had significantly higher vascularization (1093, 2186, 1333 pixel2) as the follicle was
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nearing ovulation. Hence, the mean FBF excluding three cows was 609.33 ± 89.94 pixel2 (>550
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pixel2) (Table 1). One of the cows had a regressing CL with a luteal blood flow (LBF) of 5788
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pixel2 and turbulence on the day of AI. It was present 24h following AI with increased
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vascularization (6509 pixel2) and turbulence suggestive of luteolysis. All the follicles had
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ovulated within 24 h following AI.
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The different types of CL observed on day 12 were compact CL (n=5), cavitatory CL with a
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central cavity (C) (n=1) and incompletely luteinized CL (n=6). Majority of the blood vessels
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supplying the CL on day 12 were medium (n=7) and large (n=4). There was a significant
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increase in SA, V and a 1.7 fold increase in LBF on day 21 compared to day 12 (P < 0.05). There
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was weak correlation between LBF and turbulence in blood flow to day 12 CL (r= 0.12, P <
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0.0001) and day 21 CL (r= 0.09, P < 0.0001). The blood vessels supplying the CL on day 21
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were larger (n=10) with minimal turbulence (n=2, 16.67%). Plasma P4 concentrations were
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significantly higher (P < 0.01) on day 21 compared to day 12 (Table 1).
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Conception rate in cows with normal pregnancy was 53.85% (14/26). The mean CRL, TD and
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SkD were 72.09 ± 11.56 mm, 19.65 ± 1.9 mm and 19.37 ± 3.21 mm respectively. Ossification of
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ribs was observed by 60 days (Fig 1a). Highly echogenic areas in the skull were observed around
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maxilla and mandible (Fig 1b). Clear anechoic fetal fluids and amniotic membrane was seen over
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the fetus differentiating between the allantoic and amniotic cavity (Fig 1c). The attachment of the
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umbilical cord was also observed and developing placentomes were seen as small eruptions
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protruding into the uterine lumen (Fig 1d). In the present study the mean RI and PI was 0.77 ±
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0.02 and 1.57 ± 0.3 respectively. A subjective assessment of blood flow to fetal head, body, and
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placentomes (Fig 1 d) were also performed. Normal fetal movements were observed.
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The mean LBF observed on day 60 was 5826 ± 724.1 pixel2, a two-fold increase in LBF from
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day 21 to day 60 with turbulence (Fig 1e). Filling of CL cavity can occur in many ways, however
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in the present study, hyperechogenic scar (Fig 1f) within the luteal parenchyma (substitution of
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the incompletely luteinized area present on day 21) and hyperechogenic ring surrounding the
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cavity (Fig 1g) were observed.
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3.1.2 Non-pregnant cows (n=5).
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All the follicles had ovulated within 24 h following AI except in one cow where the follicle
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persisted over 24 h with no significant increase in blood flow (200 pixel2) when compared to the
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day of AI (139 pixel2) but with a slight increase in diameter and V. A persistent CL (n=1) was
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observed on the day of AI and day 12 following AI (excluded in the calculation of average
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parameters). Mean FBF on the day of AI was 199.40 ± 68.26 pixel2 (< 250 pixel2). CL of other
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cows (n=4) regressed [partial (n=2) or complete (n=2)] by day 21. Overall, there was a 2.6 fold
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increase in blood flow nearing luteolysis with turbulence (100%) on day 21. Plasma P4
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concentration was significantly lower (P< 0.05) on day 21 when compared to day 12 (Table 1).
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There was reduction in SA, V and plasma P4 concentration.
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3.1.3 Cows with complicated pregnancy (n=6).
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Among six cows, one cow had greater amount of vascularization as it was nearing ovulation and
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the mean FBF excluding it was 286.40 ± 83.03 pixel2 (250 to 550 pixel2) (Table 1). A persistent
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CL and follicle was observed on the day of AI (V=3789.41 mm3, LBF=6364 pixel2; FBF=530
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pixel2; Fig 2a) and 24h following AI (V=2609.84 mm3, LBF=3959 pixel2; FBF=133 pixel2; Fig
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2b) in a cow. AI was performed and all the follicles had ovulated within 24 h of AI except in the
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above mentioned cow for which AI was repeated. There was no significant increase (P > 0.05) in
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mean SA, V or LBF in day 21CL compared to day 12 CL (Table 1). The size of blood vessels
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supplying CL on day 12 and 21 remained the same (small-medium) and there was turbulence
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(66.67%) in blood flow to day 21 CL. Also, there was weak positive correlation between LBF
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and turbulence to blood flow in day 12 CL (r=0.19, P < 0.01) compared to strong positive
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correlation on day 21 CL (r= 0.84, P < 0.01). Plasma P4 concentrations were significantly higher
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(P < 0.05) on day 21 compared to day 12 (Table 1).
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The different types of complications in pregnancy observed are described below. Intrauterine
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growth retardation (IUGR) was observed in two cows. The fetus on day 60 had a CRL of 46 mm
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assuming an “L” shape in one cow (Fig 2 c ) and 40 mm assuming a “C” shape in another cow
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(Fig 2 d). Ossification of the spinal column was seen in both the fetuses but ossification of the
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ribs was not evident (Fig 2 c). Amniotic membrane was evident with clear fluids. Fetal
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movements were sluggish. Late embryonic death (LED) (n=2) led to loss of embryo at 25 and 28
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days in two cows. Two cows that were declared pregnant had numerous echogenic debris
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floating within the amniotic cavity (Fig 2 e), increased endometrial thickening and placentomes
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with abnormal contour (Fig 2 f) on day 60 compared to cows with normal pregnancy (Fig 1d).
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However, both the animals sustained the fetus to term and were found to be positive for Johne’s
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disease.
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3.2 Characteristics of blood flow of multiple dominant follicles and CL in cows (n=3).
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Although there were multiple follicles, the follicle with greater V and FBF (Fig 3 a) ovulated
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within 24h and the co-dominant follicle with lower V and FBF (Fig 3 b) sustained beyond 24 h in
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two cows (n=2) (Table 2). A corpus hemorrhagicum was observed in place of the ovulated follicle
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24h following AI (n=2) and a single CL on day 12. However in one cow, the multiple dominant
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follicles had no blood supply (Fig 3 c) with remnants of previous CL (Fig 3 d). A highly
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vascularized follicle, good uterine tone and clear cervio-vaginal mucous was observed on day 12.
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However, insemination was not performed. All the cows had a CL on day 21 (Table 2).
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None of the cows with multiple dominant follicles had multiple ovulations as there was only one
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fetus on day 60 (n=2).
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4. Discussion
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The comparison between pregnant, non-pregnant and complicated pregnancy in cows was done
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on the basis of period (Day of AI, Day 12 and day 21), characteristics of follicle (SA, V, FBF)
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and CL (SA, V, LBF), presence of turbulence in blood flow to CL (day 12 vs 21), and plasma
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concentration of P4 (day 12 vs day 21) (Table 1). It was observed that there was no significant
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difference (P > 0.05) in BCS (Range: 3.8 ± 0.17 to 3.9 ± 0.05) between cows among groups
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which indicates that all the cows in this study had received adequate nutrition and the reason for
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non-pregnancy/complicated pregnancy could lie in the characteristics of the follicle and CL.
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Doppler ultrasonography was successful in evaluating functional capacity of follicle and CL to
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sustain pregnancy based on blood flow characteristics.
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Three different types of complications were observed throughout the study. Firstly, IUGR is a
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well described and extensively studied phenomenon in humans, however its implications in
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bovines have not been studied extensively. Previous reports on normal fetal characteristics have
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reported a mean CRL, TD and SkD of 60 to 70mm, 17 to 22mm, and 16 to 18mm respectively
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[17] which were comparable to the results in our study. The initial centre’s of ossification at two
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months are skull and vertebrae [18] which was also observed in cows with normal pregnancy.
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IUGR can be defined as impaired growth and development of the mammalian embryo/fetus or its
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organs during pregnancy [19] which is best determined by measuring CRL [20]. At
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approximately day 45 of gestation, the fetus loses its rudimentary embryonic shape, and its face,
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neck, limbs, and tail lengthen and become more defined [21]. A C-shaped fetus is generally
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found till 35 days and an “L” shaped fetus is found between 35 to 55 days [22]. Due to reduced
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amount of fluids, minimal ossification centre, low CRL and presence of typical embryonic shape
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(Fig 2c,d), both the cases were suggestive of IUGR.
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Secondly, LED has been defined as the death of the embryo between days 25 and 45 of gestation
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[23] which was observed in two cows. We postulate that suprabasal P4 concentration caused a
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delay in ovulation due to persistence of the regressing CL. Owing to delay in ovulation, follicular
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evacuation was prolonged thereby leading to invasion of septae into the antrum and reduced
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vascularisation (Fig 2 b) compared to day of AI (Fig 2 a). Such findings were reported in mares
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[24], but not in bovines. There was a marked delay in the rise in P4 concentrations after ovulation
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(P4 concentration of 1.45 ng/mL on day 12) which could have led to reduced production of IFN-τ
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that has a major anti-luteolytic action. This led to luteolysis, LED and return to estrus. Similar
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findings with respect to delay in ovulation and late rise in P4 leading to embryonic death has
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been reported [25,26]. Thirdly, the effect of mycobacterium paratuberculosis sp, causative agent
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of Johne’s disease, on the success of carrying a fetus to term requires further study.
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On the day of AI, there was little variation (P > 0.05) in SA, V and mean diameter between
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follicles of pregnant (PF), complicated (CPF) or non-pregnant (NPF) cows. There existed
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statistically significant variation between FBF to PF and NPF (P = 0.01) and between PF and
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CPF (P = 0.05). FBF proved to be a better parameter compared to diameter to assess the fate of
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the follicle and its impact on conception rate as there was no significant variation in follicular
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diameter among the three groups (Fig 4 a,d,g). These results agree with previous studies which
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suggested a positive relationship between the extent of blood flow to the preovulatory follicle
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and successful establishment of pregnancy in cattle [6]. This also disproved the previous notion
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that a greater follicular diameter indicates a good quality follicle. Our study indicated that it is
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important to focus on FBF irrespective of follicular diameter. In the present study cows with
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highly (> 550 pixel2), moderately (250 to 550 pixel2) or poorly (< 250 pixel2) vascularized
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follicles led to normal, complicated or non- pregnancy. Also, when the follicular blood flow was
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< 250 pixel2 at the time of AI, only 25 % of cows became pregnant or had complicated
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pregnancy, whereas a greater percentage were non-pregnant (50%.) It has been suggested that
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the control of oocyte maturation and embryo viability resides in the follicle rather than in the
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oocyte [27]. Poorly vascularized follicles produce very low quantities of E and accumulate
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greater quantities of P4 both in follicular fluid and plasma [28]. This rise in P4 has a negative
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effect on LH pulse frequency [29], thereby delaying ovulation and affecting oocyte maturation
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which eventually leads to return to estrus. However, moderately vascularized follicles could
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trigger significant production of E (but lower than highly vascularized follicles) which might
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regulate LH pulses. During ovulation, the invasion of thecal cells and blood vessels into the
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granulosa cells is necessary for production of early pregnancy factor (EPF) and transformation
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into a good quality CL along with an early switch in P4 production [26]. Therefore, moderately
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vascularized follicles could produce less EPF leading to complications in pregnancy compared to
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highly vascularized follicles (> 550 pixel2) which may be the limiting step which decides
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whether a pregnancy should proceed towards normalcy or complication.
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On day 12, there was no significant variation (P > 0.05) between mean diameter, SA, V and LBF
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to CL or plasma P4 concentration among cows that remained pregnant (PCL), non-pregnant
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(NPCL) or that had a complicated pregnancy (CPCL). There was weak correlation between LBF
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and turbulence in blood flow in both PCL (r= 0.12, P < 0.0001) and CPCL (r=0.19, P < 0.01).
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None of the parameters assessed on day 12 were useful in differentiating among the three groups
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(Fig 4 b,e,h). However, day 21 CL was a better indicator as there were significant differences on
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comparison of parameters between the three groups. Except for SA between PCL and CPCL
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(P>0.05), there was statistically significant variation between SA, V and P4 concentration
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between PCL, CPCL and NPCL (Table 1). LBF alone was not beneficial in differentiating
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among the three groups (P > 0.05), but interesting results were obtained on evaluation of LBF
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along with turbulence to blood flow in day 21 CL (Fig 4 c,f,i). There was weak correlation
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between LBF to PCL and turbulence to blood flow (r= 0.09, P < 0.0001), but there was a strong
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positive correlation between LBF to CPCL and turbulence to blood flow (r= 0.84, P < 0.01).
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Turbulence to day 21 CL was observed in all NPCL (Table 1). It was also observed that there
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was significant increase in LBF (1.7 fold) and V (P < 0.05) from day 12 to 21 in pregnant cows,
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but there was no significant increase in LBF and V (P > 0.05) from day 12 to 21 in cows with
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complicated pregnancy. A 2.6 fold increase in LBF and 3.7 fold decrease in V in NPCL with
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significant turbulence was observed on day 21 when compared to day 12 which indicated the
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start of luteolysis. Thus assessment of turbulence and increase in LBF from 12 to 21 days post AI
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can give a fair indication of the fate of a CL. Assessment of turbulence and LBF can be used to
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predict luteolysis which can save time and increase profitability in a dairy farm.
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A delayed rise in P4 during early pregnancy (as seen on comparison of pregnant cows and cows
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with complicated pregnancy in Table 1) suggests luteal inadequacy and it has been reported that
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cows that underwent an earlier rise in P4 had embryos that were further developed and produced
311
more of the antiluteolytic protein, interferon –tau (IFN-τ) by day 16 than cows that had a delayed
312
rise in concentrations of P4 [25]. IFN-τ along with P4 secreted by CL exerts their antiluteolytic
313
effect by modifying oxytocin receptors inhibiting the synthesis from arachidonic acid and
314
subsequent release of PGF2α [30]. The maximum secretion of IFN-τ occurs between days 16-19
315
of gestation [31]. The results from previous studies indicate that low P4 concentrations during an
316
estrus cycle have a delayed stimulatory effect on uterine responsiveness to oxytocin during the
317
late luteal phase of the subsequent cycle [32]. The resulting increase in PGF2α secretion may
318
interfere with luteal maintenance during the early stages of pregnancy [32]. This could suggest
319
that the suboptimal P4 in cows with complicated pregnancy (day 12; Table 1) was insufficient in
320
preventing the release of PGF2α, thereby affecting the growth and survival of the fetus.
321
Turbulence in blood flow, though subjective, was minimum in PCL compared to NPCL (P <
322
0.01) and CPCL (P < 0.05). Thus, a minimal release of PGF2α might be responsible for the
323
increase in turbulence in day 21 CPCL in cows which may or may not cause death of the fetus,
324
but the success in carrying it to term may be hampered and requires further study.
325
These findings suggested that LBF, turbulence to CL and plasma P4 concentration on day 21
326
were valuable indicators reflecting the functional capacity of the CL between different groups.
327
These results agree with previous studies which indicate that LBF is a reliable indicator of luteal
328
status [33]. However, our study adds turbulence to blood flow as a new parameter along with
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LBF. Plasma P4 concentration remains the gold standard for evaluation of luteal function [34].
330
But P4-based diagnosis is time-consuming, hence difficult to obtain results immediately after
331
sampling. Therefore colour Doppler ultrasonography can be a useful cow-side tool for immediate
332
assessment of ovarian structures and prediction of functional status.
333
CL at the time of AI was observed in all groups. However, LBF determined the fate of the CL. A
334
regressing CL present on the day of AI (Fig 5a) had increased LBF and turbulence 24h following
335
AI (Fig 5b) suggesting that it was undergoing complete luteolysis and the functional capacity of
336
the CL in producing P4 was very low leading to a normal pregnancy. However, a persistent CL
337
on the day of AI (Fig 5c) had decreased LBF and absence of turbulence 24h following AI (Fig
338
5d). The prolonged life span of the CL caused delay in ovulation of follicle due to the negative
339
impact of P4 produced by the CL on LH pulses attributing to LED. The negative effects of P4
340
have also been reported in several other studies [35,36]. These findings suggest that assessment
341
of vascularization to regressing CL revealed different patterns of blood flow which can be
342
effective in predicting the pattern of regression and effect on pregnancy.
343
Although two cows had multiple dominant follicles, multiple ovulations were not observed
344
because there was a difference in vascularization between the multiple dominant follicles and
345
only the highly vascularized follicle ovulated whereas the co-dominant follicle persisted and
346
regressed at a later stage. Thus it can be concluded that Doppler ultrasonography can be a useful
347
aid in detection of multiple ovulation which can lead to twins.
348
5. Conclusion
349
Assessment of vascularization to follicle (FBF) on the day of AI and CL (LBF and turbulence to
350
blood flow) during early pregnancy using Doppler ultrasonography proved to be a successful
351
predictor of fertility. Cows with a highly vascularized follicle (> 550 pixel2) underwent a normal
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pregnancy, whereas those that had moderately (250 to 550 pixel2) and poorly (< 250 pixel2)
353
vascularized follicle experienced complicated pregnancy or remained non-pregnant, respectively.
354
On day 21, LBF alone was not beneficial in differentiating among the three groups, but
355
assessment of LBF along with turbulence to blood flow in day 21 CL proved highly valuable due
356
to an increased turbulence in CPCL (66.67%) compared to PCL (16.67%). Assessment of
357
turbulence and LBF can also be used to predict luteolysis in CL.
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Acknowledgements
360
The authors thank the faculty and staff of the Department of Veterinary Gynaecology and
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Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) for their
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help and advice. We show our sincere gratitude to the working staff and faculty at the Dairy
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Farm Complex and dairy farms for their continued support.
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365 References
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364
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366 [1] Breuel KF, Lewis PE, Schrick FN, Lishman AW, Inskeep EK, Butcher RL. Factors affecting 367 fertility in the postpartum cow: Role of oocyte and follicle in conception rate. Biol Reprod 368 1993; 48: 655-61.
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369 [2] Brannstrom M, Zackrisson U, Hagstrom HG, Josefsson B, Hellberg P, Granberg S, Collins 370 WP, Bourne T. 1998. Preovulatory changes of blood flow in different regions of the human 371 follicle. Fertil Steril 69: 435-42. 372 [3] Ginther OJ. Arteries and Hemodynamics. In: Ginther OJ. Ultrasonic Imaging and Animal 373 Reproduction: Color-Doppler Ultrasonography, Wisconsin: Equiservices Publishing, Cross 374 Plains; 2007, p. 7-24. 375 [4] Coulam CB, Goodman C, Rinehart JS. Colour Doppler indices of follicular blood flow as 376 predictors of pregnancy after in-vitro fertilization and embryo transfer. Hum Reprod 1999; 14: 377 1979–82.
16
ACCEPTED MANUSCRIPT
378 [5] Huey S, Abuhamad A, Barroso G, Hsu MI, Kolm P, Mayer J, Oehninger S. Perifollicular blood 379 flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental 380 competence in in vitro fertilization. Fertil Steril 1999; 72: 707–12.
RI PT
381 [6] Siddiqui MAR, Almamun M, Ginther OJ. Blood flow in the wall of the preovulatory follicle 382 and its relationship to pregnancy establishment in heifers. Anim Reprod Sci 2009; 113: 287– 383 92. 384 [7] Kastelic JP, Bergfelt DR, Ginther OJ. Relationship between ultrasonic assessment of the corpus 385 luteum and plasma progesterone concentration in heifers. Theriogenology 1990a; 33: 1269386 78.
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387 [8] Bergfelt DR, Ginther OJ. Ovarian and embryo dynamics in horses versus ponies. J Equine Vet 388 Sci 1996; 16: 66-72.
M AN U
389 [9] Kawakami S, Shida T, Mutoh M, Kohmoto H, Onhchi T. Relation between luteal regression 390 and so-called counter current mechanisms in the cow: Verification from PGF2α 391 concentrations in arterial, uterine venous and jugular venous blood following PGF2α loading. 392 J Reprod Dev 1995; 41: 219–23. 393 [10] Acosta T J, Yoshizawa N, Ohtani M, Miyamoto A. Local changes in blood flow within the 394 early and midcycle corpus luteum after prostaglandin F(2 alpha) injection in the cow. Biol 395 Reprod 2002; 66: 651–8.
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396 [11] Carrière PD, Gnemmi G, Descôteaux L, Matsui M, Miyamoto A, Colloton J. Bovine ovary. 397 In: Des Côteaux L, Colloton J and Gnemmi G. Practical atlas of ruminant and camelid 398 reproductive ultrasonography, United Kingdom: Wiley-Blackwell publishers; 2010, p. 35-60. 399 [12] Ginther OJ, Utt MD. Doppler Ultrasound in Equine Reproduction: Principles, Techniques, 400 and Potential. J Equine Vet Sci 2004; 24: 516-26.
Imaging
#4.
EP
401 [13]Kisslo JA, Adams DB. Doppler Color Flow 402 http://cardioland.org/Echo/doppler04.pdf [accessed on 05.02.17].
403 [14] Pierson RA, Ginther OJ. Ultrasonography of the bovine ovary. Theriogenology 1984; 21: 404 495–504.
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405 [15] Ghuman SPS, Dadarwal D, Honparkhe M, Singh J, Dhaliwal GS. Production of polyclonal 406 antiserum against progesterone for radioi-mmunoassay. Indian Vet J 2009; 86: 909-11. 407 [16] SPSS 16.0, 2007: Command Syntax Reference. SPSS Inc©, South Wacker Drive, Chicago. 408 [17] Kahn W. Sonographic fetometry in the bovine . Theriogenology 1989; 31: 1105-21. 409 [18] Gjesdal F. Age determination of bovine foetuses. Acta Vet Scand 1969; 10: 197-218. 410 [19] Wu G, Bazer FW, Wallace JM, Spencer TE. Intrauterine growth retardation: Implications for 411 the animal species. J Anim Sci 2006; 84: 2316-37. 412 [20] Mandruzzato G. Intrauterine growth restriction (IUGR): Guidelines for definition, recognition 413 and management. Arch Perin Med 2008; 14: 7-8. 17
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414 [21] Descôteaux L, Colloton J, Gayrard V, Picard-Hagen N. Bovine pregnancy, In: Des Côteaux L, 415 Colloton J and Gnemmi G. Practical atlas of ruminant and camelid reproductive 416 ultrasonography, United Kingdom: Wiley-Blackwell publishers; 2010, p. 81-99.
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417 [22] Kolour AK, Batavani RA, Ardabili FF. Preliminary observations on the effect of parity on 418 first day ultrasonic detection of embryo and its organs in bovine. J Vet Med A Physiol Pathol 419 Clin Med 2005; 52: 74-7. 420 [23] Committee on Bovine Reproductive Nomenclature. Recommendations for standardising 421 bovine reproductive terms. Cornell Veterinary 1972; 216-37.
SC
422 [24] Ginther OJ, Gastal EL, Gastal MO. Spatial Relationships between Serrated Granulosa and 423 Vascularity of the Preovulatory Follicle and Developing Corpus Luteum. J Equine Vet Sci 424 2007; 27: 20-7.
M AN U
425 [25] Mann GE, Lamming GE. Relationship between maternal endocrine environment, early 426 embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 2001; 427 121: 175-80. 428 [26] Hafez ESE, Hafez B. Folliculogenesis, egg maturation and ovulation. In: Hafez ESE, Hafez 429 B. Reproduction in Farm Animals, Pennsylvania: Lipincott Williams and Wilkins; 2000, p.79. 430 [27] Moor RM, Dai Y, Lee C, Fulka JJr. Oocyte maturation and embryonic failure. Human 431 Reproduction 1998; 4: 223–36 (Update).
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432 [28] Varughese EE. Assessment of the vascularization of ovarian structures and their correlation 433 with fertility in dairy animals, M.V.Sc Thesis, Guru Angad Dev Veterinary and Animal 434 Sciences University, Ludhiana, Punjab, India. 2012. 435 [29] Adams GP, Matteri RL, Ginther OJ. Effect of progesterone on ovarian follicles, emergence of 436 follicular waves and circulating follicle-stimulating hormone in heifers. J Reprod Fertil 1992; 437 95: 627–40.
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438 [30] Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferon-τ production 439 and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999; 54: 317-28 440 (Supple).
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441 [31] Bartol FF, Roberts RM, Bazer FW. Characetrization of proteins produced by peri-attachment 442 bovine conceptuses. Biol Rerod 1985; 32: 681-93. 443 [32] Wolfenson D. Factors associated with low progesterone concentrations and their relation to 444 low fertility of lactating dairy cows. Israel J Vet Med 2006; 61. 445 [33] Herzog K, Brockhan-Ludemann M, Kaske M, Beindorff N, Paul V, Niemann H, Bollwein H. 446 Luteal blood flow is a more appropriate indicator for luteal functionduring the bovine estrous 447 cycle than luteal size. Theriogenology 2010; 73: 691–7. 448 [34] Kastelic JP, Pierson RA, Ginther OJ. Ultrasonic morphology of corpora lutea and central 449 luteal cavities during the estrous cycle and early pregnancy in heifers. Theriogenology 1990b; 450 34: 487–98
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451 [35] Gustafsson H, Larsson K, Kindahl H, Madej A. Sequential endocrine changes and behavious 452 during estrus and metestrus in repeat breeder and virgin heifers. Anim Reprod Sci 1986; 19: 453 261-73.
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454 [36] Albihn A, Gustafsson H, Hurst M, Rodriguez-Martinez H. Embryonic ability to prolong the 455 interestrus interval in virgin and repeat breeding heifers. Anim Reprod Sci 1991; 26: 193-210.
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Table 1
Characteristics of CL on day 12
SA (mm2)
V (mm3)
FBF (pixel2)
Mean diameter (mm)
SA (mm2)
V (mm3)
LBF (pixel2)
15.98 ± 1.73
164.92 ± 17.67
2011.23 ± 306.88
609.33 ± 89.94a*a
19.06 ± 1.22
252.07 ± 14.54
3429.48 ± 296.37g
1868.67 ± 121.83g
14.31 ± 2.23
117.52 ± 18.76
1352.38 ± 298.94
199.40 ± 68.26b
20.35 ± 2.55
Complicated Pregnancy (n=6)
14.25 ± 1.35
134.11 ± 19.61
1410.87 ± 240.72
286.40 ± 83.03c
18.82 ± 1.99
256.67 ± 34.56
228.13 ± 30.11
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NonPregnant (n=5)
Characteristics of CL on day 21
P4 (ng/ mL)
Mean diameter (mm)
SA (mm2)
V (mm3)
LBF (pixel2)
P4 (ng/ mL)
T (% of cases )
2.19 ± 0.11
21.68 ± 2.14
304.42 ± 17.94d
4947.95 ± 286.43a*
3124.50 ± 243.34f
3.36 ± 0.23a*
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Mean diameter (mm)
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Pregnant (n=12)
Characteristics of follicle
df
bc
d
3464.43 ± 819.29
1265.00 ± 174.46
2.30 ± 0.24
12.37 ± 0.58
109.30 ± 7.23e
946.58 ± 124.16e
3268.50 ± 772.50
0.79 ± 0.21be
100 a
3191.47 ± 485.17
1961.17 ± 418.04g
1.83 ± 0.21
19.80 ± 1.62
260.20 ± 14.80d
3722.37 ± 284.16cd
2771.83 ± 348.45f
2.51 ± 0.20ac
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Comparison between pregnant, non-pregnant and complicated pregnancy in cows on the basis of characteristics of follicle, CL, turbulence to CL and plasma progesterone (P4) concentration (excluding cows with multiple dominant follicles).
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a represents significantly (P ≤ 0.01) higher than b within columns between similar variables. a* represents significantly (P ≤ 0.05) higher than c within columns between similar variables. d represents significantly (P ≤ 0.0001) higher than e within columns between similar variables. f represents significantly (P ≤ 0.05) higher than g within rows between similar variables.
20
a*
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Table 2
Animal No.
Characteristics of follicles on day of AI
FBF 24 h of AI
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Morphological evaluation and characteristics of blood flow of multiple dominant follicles on the day of artificial insemination (AI), 24h following AI and CL on day 12 and 21 following AI in cows.
Characteristics of CL on day 12
2*.
3^.
SA (mm2)
176.71
2506.48
821
-
84.95
837.20
243
400
211.24
2489.79
515
-
141.03
1727.94
64
250
88.25
781.56
0
0
88.25
646.41
0
0
LBF (pixel2)
V (mm3)
Characteristics of cavity
P4 (ng/ mL)
SA (mm2)
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FBF (pixel2)
V (mm3)
LBF (pixel2)
V (mm3)
Type
-
-
1.70
350.67
4955.64
2822
Presence of turbulence
Characteristics of cavity
P4 (ng/ mL)
V (mm3)
Type
-
-
-
3.0
263.02
3765.67
1987
593.96
9513.48
2146
1363.32
C
2.65
650.78
9899.65
3022.12
-
874.32
C
3.3
-
-
-
-
-
0.33
543.25
9586.5
2610
+
1062.34
C
1.7
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1*.
V (mm3)
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SA (mm2)
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(pixel2)
Characteristics of CL on day 21
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*1st follicle ovulated and the 2nd follicle was present ^Cow came in heat on day 12 with clear and thick estrous discharge and strong uterine tone, but insemination was not performed.
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502
Figure legends
503
Fig. 1. Pregnancy diagnosis on day 60 in cows with normal pregnancy (a) Ossification of ribs and spinal column (yellow arrows), (b) Ossification centres around maxillary and mandibular bones, (c) An intact amniotic membrane overlying the fetus (red arrow), (d) Placentomes were seen as small eruptions and offset indicates the blood flow to placentomes, (e) LBF on day 60 indicating copious blood supply with mild turbulence (yellow arrow) suggestive of greater velocity in blood flow to supply the pregnant CL, (f) An incompletely luteinized CL of day 21 replaced by a hyperechogenic scar (red arrow) on day 60, (g) Persistence of central cavity in day 60 CL with development of a hyperechogenic ring (orange arrow) around the cavity,
513 514 515 516 517 518 519 520 521
Fig. 2. Blood flow to follicle on day of artificial insemination (AI) and pregnancy diagnosis on day 60 in cows with complicated pregnancy (a) Blood flow to follicle on day of AI (530 pixel2), (b) Prolonged sustenance of the follicle with reduction in blood flow (133 pixel2) and initiation of formation of septae (white arrow) within the follicular antrum, 24 hrs following AI, (c) L-shaped fetus with a crown-rump length (CRL) of 46 mm and ossification of spinal column (black arrow) with no evidence of ossification of ribs, (d) Doppler on C-shaped fetus with a CRL of 40 mm showing blood supply to fetus. (e) Echogenic debris within amniotic cavity, (f) Increased endometrial thickening (red arrow) and placentomes with abnormal contour.
522 523 524 525 526
Fig. 3. Characteristics of multiple dominant follicle’s. (a) Follicle with greater volume and vascularization on the day of AI (515 pixel2) (b) Co-dominant follicle with lower volume and vascularization on the day of AI (64 pixel2), (c) Multiple follicle’s in Cow 3 with absence of blood supply on the day of AI, (d) Remnants of CL were observed on the day of AI which is evident from the increased vascularization.
532 533 534 535 536 537
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Fig. 4. Assessment of vascularization to follicle and CL in three groups. Pregnant cow - (a) Follicular blood flow (FBF) on the day of AI (934 pixel2), (b) Day 12 CL (1758 pixel2), (c) Day 21 CL (3948 pixel2). Note the increase in size of blood vessels (orange arrow) on day 21 compared to day 12 and absence of turbulence in blood flow on both day 12 and day 21 CL.
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504 505 506 507 508 509 510 511 512
Complicated pregnancy (Intrauterine growth retardation) (d) FBF on the day of AI (340 pixel2), (e) Day 12 CL (2716 pixel2), (f) Day 21 CL (3209 pixel2). Turbulence was present in both day 12 and 21 CL (yellow arrow). There was no considerable increase in size of the CL or blood vessels in day 21 CL compared to day 12 CL. Non-pregnant cow– (g) FBF on the day of AI (139 pixel2), (h) Day 12 CL with an eccentric cavity and absence of turbulence (1362 pixel2), (i) Day 21 CL (bottom) with
22
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a significant increase in vascularization (4041 pixel2), turbulence (white arrow), reduction in size indicative of luteolysis and presence of follicle (top).
540 541 542 543 544 545 546
Fig. 5. Luteolysis and fate of corpus luteum (CL) assessed by vascularization and turbulence in luteal blood flow (LBF). (a) CL on the day of AI with LBF (5788 pixel2) and minimal turbulence, (b) CL with increased LBF (6509 pixel2) and greater turbulence (yellow arrow) 24 hrs following AI which suggests that CL might undergo complete luteolysis, (c) CL on day of AI with good LBF (6364 pixel2) and moderate turbulence (yellow arrow), (d) CL 24 hrs following AI with decreased LBF (3959 pixel2) and absence of turbulence which suggests a persistent CL.
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Highlights Predict pregnancy rate based on vascularization to follicle and corpus luteum (CL).
•
Cows with a highly vascularized follicle (> 550 pixel2) underwent a normal pregnancy.
•
Moderately (250 to 550 pixel2) and poorly (< 250 pixel2) vascularized follicle
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experienced complicated pregnancy or remained non-pregnant, respectively. •
Assessment of luteal blood flow along with turbulence to blood flow in day 21 CL
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pregnant cows (16.67%).
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Increased turbulence in CL of complicated pregnant cows (66.67%) compared to CL of
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proved highly valuable.
S0093-691X(17)30373-4
DOI:
10.1016/j.theriogenology.2017.07.042
Reference:
THE 14208
To appear in:
Theriogenology
Received Date: 9 May 2017 Revised Date:
21 July 2017
Accepted Date: 27 July 2017
Please cite this article as: Varughese EE, Brar PS, Ghuman SS, Vascularization to preovulatory follicle and corpus luteum-a valuable predictor of fertility in dairy cows, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.07.042. 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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REVISED
Vascularization to preovulatory follicle and corpus luteum- a valuable predictor of fertility
2
in dairy cows
3
Emy E. Varughese*, Parkash S. Brar, Sarvpreet S. Ghuman
4 5
Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, India
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* Corresponding author: Tel: +1 614-592-5900
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Email: [email protected]
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Present address: 5300 Riverside Dr, Upper Arlington, Ohio, USA 43220
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Abstract
The aim of the present study was to predict pregnancy rate based on vascularization to follicle
11
and corpus luteum (CL). 26 Holstein Friesian cows were synchronized using Ovsynch protocol.
12
On day 10 of the protocol, vascularization and morphological characteristics [sectional area
13
(SA), volume (V) and mean diameter] of follicle was assessed and animals underwent artificial
14
insemination (AI). Morphological evaluation and vascularization to CL was assessed on day 12
15
and 21 following AI and blood samples were obtained for estimation of plasma progesterone
16
(P4). Pregnancy diagnosis was performed on day 60 of AI and was classified as normal,
17
complicated and non-pregnant. The overall conception rate was 76.92 % (20/26); normal
18
pregnancy was 53.85% (14/26). Complications observed in pregnancy were intrauterine growth
19
retardation, late embryonic death and infection. Cows with a highly vascularized follicle (> 550
20
pixel2) underwent a normal pregnancy, whereas those that had moderately (250 to 550 pixel2)
21
and poorly (< 250 pixel2) vascularized follicle experienced complicated pregnancy or remained
22
non-pregnant, respectively. On day 12, there was no significant variation (P > 0.05) between
23
mean diameter, SA, V, luteal blood flow (LBF) or plasma P4 concentration among CL of cows
24
that remained pregnant (PCL), non-pregnant (NPCL) or that had a complicated pregnancy
25
(CPCL). LBF alone was not beneficial in differentiating among the three groups (P > 0.05), but
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assessment of LBF along with turbulence to blood flow in day 21 CL proved highly valuable due
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to an increased turbulence in CPCL (66.67%) compared to PCL (16.67%). Assessment of
28
turbulence and LBF on day 12 and 21 can also be used to predict luteolysis with accuracy.
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Keywords: Doppler ultrasonography; Follicle; Corpus luteum; Blood flow; Pregnancy.
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1. Introduction
32
A calf per animal per year is the most favorable realization of every farmer/dairy enterprise. A
33
successful pregnancy involves several important stages. Firstly, an ovarian follicle must develop
34
and ovulate an oocyte capable of being fertilized and undergo embryonic development.
35
Secondly, the oviductal and uterine environments must be suitable for gamete transport,
36
fertilization, and subsequent embryonic development. Finally, the corpus luteum (CL) must
37
function for a sufficient period of time for maternal recognition of pregnancy and maintain
38
gestation [1]. Hemodynamic changes are involved in the cyclic remodeling of ovarian tissue that
39
occurs during final follicular growth, ovulation and new CL development [2] during which
40
angiogenesis plays a crucial role in the maintenance of ovarian structures.
41
Transrectal Doppler ultrasonography has been utilized increasingly for research and clinical
42
studies of ovarian hemodynamics involving follicle and CL in large farm animals [3]. Blood
43
flow determinations of individual preovulatory follicles provide an important index on the
44
intrafollicular environment and may predict the developmental competence of the corresponding
45
oocyte [4,5]. A study on 39 Holstein heifers proved that blood flow at the time of artificial
46
insemination (AI) was greater in the preovulatory follicle wall of heifers that became pregnant
47
than in heifers that did not [6]. Relationships between the echogenicity of CL and progesterone
48
(P4) concentrations have already been made with conventional gray-scale ultrasound in heifers
49
[7] and in mares [8] and an increased blood flow has been proposed to augment the transfer of
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the embryonic signal for CL maintenance and for maternal recognition through the utero-ovarian
51
pathway [9].
52
Therefore, assessment of blood flow to ovarian structures (follicle and CL) has been shown to
53
give preliminary information regarding pregnancy rate [6]. However continuous assessment of
54
follicle, CL and sustenance of pregnancy has not been studied. Early prediction of nature of
55
pregnancy based on blood flow to follicle and CL will prove to be a boon for the dairy industry.
56
Infertility and complications in pregnancy remains to contribute significantly to reduced
57
conception rates in dairy farms. Since follicle and CL are ovarian structures easily visualized by
58
ultrasonography and indispensable for a successful pregnancy, our study aimed at assessing
59
blood flow to follicle and CL to predict fertility in dairy cows.
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2. Materials and methods
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2.1 Animals
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The study was conducted on twenty-six healthy, reproductively normal and regular cycling
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postpartum Holstein Friesian cows (body weight: 300 to 350 kg, body condition score (BCS): 3.5
65
to 4, parity: 2 to 3). The cows were housed in an organized dairy farm in semi-loose housing
66
system and were reared on green fodder (30 to 40 kg), wheat straw (2 kg), concentrate feed,
67
mineral mixture and ad libitum drinking water.
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2.2 Ovulation synchronization (Ovsynch)
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Irrespective of the stage of estrus cycle, the cows were subjected to an ovulation synchronization
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protocol. Cows were treated with a GnRH analogue (Buserelin acetate, ReceptalTM, Intervet,
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India, im) on day 0 (20 µg) and day 9 (10 µg) along with a synthetic prostaglandin F2α analogue
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(Cloprostenol Sodium, VetmateTM, Vetcare, Bangluru, India, 500 µg im) on day 7. 3
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2.3 Study design
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On the day of estrus (Day 10, of Ovsynch protocol), transrectal examination and ultrasonography
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were used to locate and measure the size of follicle respectively. Colour flow mode (CFM) was
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used to assess the blood supply to the follicle, and timed AI was performed. Blood flow of the
78
follicle was reassessed on day 11 (24hrs following AI), and AI was repeated if the follicle had
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not ovulated. Size and blood flow of CL was assessed on day 12 and 21 following AI
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(represented as day 12 and 21 respectively). Blood samples were collected on day 12 and 21 for
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estimation of P4. Pregnancy diagnosis was performed in all the animals on day 60 of AI using
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ultrasonography.
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2.4 Brightness mode (B-mode) and Doppler ultrasonography
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Transrectal ultrasonography was carried out using a battery operated B-mode ultrasound scanner
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equipped with a 7.5 MHz, linear-array transducer (Exago, ECM - Noveko International
87
Inc., Angoulème, France) for assessing the size of follicle/CL. The frozen optimal scan images
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were used to determine the diameter (average of maximum length and transverse diameter) with
89
the help of built-in, on-screen callipers. The volume (V) of the follicle and CL were estimated
90
using the following equation for a modified prolate ellipsoid: V = 0.523 × A × B2, in which A
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represents the maximum length and B represents the transverse diameter [10]. CL were described
92
as CL with a single (central or eccentric)/multiple cavities or CL with incomplete luteinization of
93
the follicular wall [11]. If a CL contained a cavity, volume of the cavity was determined and
94
subtracted from the calculated CL volume except in CL with incomplete luteinization. In
95
addition, the image obtained in a vertical plane from the apex to the base of the follicle/CL,
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designated as the overall image was used to determine the sectional area (SA) of the follicle/CL:
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SA= π/4 × (SD)2, where SD is the sectional diameter [10].
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Thereafter, color flow mode (4000 Hz pulse repetition frequency and 27.5 dB gain) of the
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scanner was used for blood flow mapping of follicle/CL. The red color indicated blood-flow
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toward the transducer’s face, and blue color indicated blood-flow away from the transducer’s
101
face [12]. When the transducer was placed close to the follicle/CL, the blood flow captured by
102
the transducer appeared as different color intensities on the monitor. As the velocity of the flow
103
increased, the color intensity increased. Forward flow with turbulence resulted in yellow and
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backward flow with turbulence resulted in cyan (blue-green) [13]. Transducer was positioned at
105
the maximal diameter of the follicle/CL to achieve the maximal number of color pixels in the
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recorded image. The blood vessels supplying the structures were evaluated based on their
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diameter into small (< 3mm), medium (3-6mm) and large (> 6mm).
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2.5 Quantification of blood flow
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Colored spots or pixel aggregates were selected from the images, extracted, and saved using
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Adobe Photoshop software (Adobe ® Photoshop ® CS3 Extended, Version 10.0, USA). Image J
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(Image J 1.45s, USA) was used to calculate total number of colored pixels, expressed as pixel2.
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2.6 Pregnancy diagnosis
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Pregnancy was confirmed at Day 60. Fetus appeared as an echogenic structure inside a non-
116
echogenic structure [14] using B-mode ultrasonography. Once the fetus was detected, image was
117
frozen and crown-rump length (CRL, a straight line between the fetal crown and the origin of the
118
tail), trunk diameter (TD, at level of umbilical cord attachment) and skull diameter (SkD, the
119
widest diameter) were measured and the image was recorded in a few normal and other cases.
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Blood flow velocity wave forms of the middle uterine artery were recorded in those cases which
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were suspected for fetal growth retardation/abnormalities and in normal cases for comparison.
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The middle uterine artery was located using CFM and switched to Pulsed-wave mode (4000Hz
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pulse repetition frequency, 12 dB gain, 50% power and 20 to 60 degrees Doppler angle) for
124
determination of waveform. The waveform was evaluated for Resistance Index (RI) and
125
Pulsatility Index (PI). A higher resistance or RI indicated lower perfusion to the particular organ
126
and a high PI indicated decreased perfusion to distal tissues [12].
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Based on the blood supply of follicle/CL, fetal characteristics and blood flow velocity
128
waveforms, pregnancy was classified as normal, complicated and non-pregnant.
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2.7 Blood sampling
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Jugular vein blood samples (10 ml) were collected in heparinized vaccutainer vials prior to
132
ultrasonography on day 12 and 21. Plasma was separated immediately after blood collection by
133
centrifugation at 3000 X g for 15 min. The plasma aliquots were stored at −20°C for estimation
134
of P4.
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2.8 Estimation of P4
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Plasma P4 was assayed with a solid-phase radioimmunoassay using antisera raised in our
138
laboratory [15]. Sensitivity of the assay was 0.1 ng/ml; intra- and inter-assay variation
139
coefficients were 6.2% and 9.5%, respectively.
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2.9 Statistical Analysis
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Numerical data is expressed as Mean ± SEM. Statistical analysis was performed using SPSS 16
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[16]. Pearson’s correlation coefficient (r) tested the correlation between variables. One-way
144
ANOVA was used to compare means within variables. Post hoc test (Tukey HSD) was used to
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3. Results
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Pregnancy diagnosis on day 60 revealed an overall conception rate of 76.92 % (20/26).
148
3.1 Characteristics of blood flow of follicle and CL and its correlation with plasma concentration
149
of P4 in:
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3.1.1 Pregnant cows (n=12).
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Follicular blood flow (FBF) on the day of AI was 841.33 ± 155.22 pixel2 (n=12). However, three
153
cows had significantly higher vascularization (1093, 2186, 1333 pixel2) as the follicle was
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nearing ovulation. Hence, the mean FBF excluding three cows was 609.33 ± 89.94 pixel2 (>550
155
pixel2) (Table 1). One of the cows had a regressing CL with a luteal blood flow (LBF) of 5788
156
pixel2 and turbulence on the day of AI. It was present 24h following AI with increased
157
vascularization (6509 pixel2) and turbulence suggestive of luteolysis. All the follicles had
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ovulated within 24 h following AI.
159
The different types of CL observed on day 12 were compact CL (n=5), cavitatory CL with a
160
central cavity (C) (n=1) and incompletely luteinized CL (n=6). Majority of the blood vessels
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supplying the CL on day 12 were medium (n=7) and large (n=4). There was a significant
162
increase in SA, V and a 1.7 fold increase in LBF on day 21 compared to day 12 (P < 0.05). There
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was weak correlation between LBF and turbulence in blood flow to day 12 CL (r= 0.12, P <
164
0.0001) and day 21 CL (r= 0.09, P < 0.0001). The blood vessels supplying the CL on day 21
165
were larger (n=10) with minimal turbulence (n=2, 16.67%). Plasma P4 concentrations were
166
significantly higher (P < 0.01) on day 21 compared to day 12 (Table 1).
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Conception rate in cows with normal pregnancy was 53.85% (14/26). The mean CRL, TD and
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SkD were 72.09 ± 11.56 mm, 19.65 ± 1.9 mm and 19.37 ± 3.21 mm respectively. Ossification of
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ribs was observed by 60 days (Fig 1a). Highly echogenic areas in the skull were observed around
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maxilla and mandible (Fig 1b). Clear anechoic fetal fluids and amniotic membrane was seen over
171
the fetus differentiating between the allantoic and amniotic cavity (Fig 1c). The attachment of the
172
umbilical cord was also observed and developing placentomes were seen as small eruptions
173
protruding into the uterine lumen (Fig 1d). In the present study the mean RI and PI was 0.77 ±
174
0.02 and 1.57 ± 0.3 respectively. A subjective assessment of blood flow to fetal head, body, and
175
placentomes (Fig 1 d) were also performed. Normal fetal movements were observed.
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The mean LBF observed on day 60 was 5826 ± 724.1 pixel2, a two-fold increase in LBF from
177
day 21 to day 60 with turbulence (Fig 1e). Filling of CL cavity can occur in many ways, however
178
in the present study, hyperechogenic scar (Fig 1f) within the luteal parenchyma (substitution of
179
the incompletely luteinized area present on day 21) and hyperechogenic ring surrounding the
180
cavity (Fig 1g) were observed.
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3.1.2 Non-pregnant cows (n=5).
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All the follicles had ovulated within 24 h following AI except in one cow where the follicle
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persisted over 24 h with no significant increase in blood flow (200 pixel2) when compared to the
185
day of AI (139 pixel2) but with a slight increase in diameter and V. A persistent CL (n=1) was
186
observed on the day of AI and day 12 following AI (excluded in the calculation of average
187
parameters). Mean FBF on the day of AI was 199.40 ± 68.26 pixel2 (< 250 pixel2). CL of other
188
cows (n=4) regressed [partial (n=2) or complete (n=2)] by day 21. Overall, there was a 2.6 fold
189
increase in blood flow nearing luteolysis with turbulence (100%) on day 21. Plasma P4
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concentration was significantly lower (P< 0.05) on day 21 when compared to day 12 (Table 1).
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There was reduction in SA, V and plasma P4 concentration.
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3.1.3 Cows with complicated pregnancy (n=6).
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Among six cows, one cow had greater amount of vascularization as it was nearing ovulation and
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the mean FBF excluding it was 286.40 ± 83.03 pixel2 (250 to 550 pixel2) (Table 1). A persistent
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CL and follicle was observed on the day of AI (V=3789.41 mm3, LBF=6364 pixel2; FBF=530
196
pixel2; Fig 2a) and 24h following AI (V=2609.84 mm3, LBF=3959 pixel2; FBF=133 pixel2; Fig
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2b) in a cow. AI was performed and all the follicles had ovulated within 24 h of AI except in the
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above mentioned cow for which AI was repeated. There was no significant increase (P > 0.05) in
199
mean SA, V or LBF in day 21CL compared to day 12 CL (Table 1). The size of blood vessels
200
supplying CL on day 12 and 21 remained the same (small-medium) and there was turbulence
201
(66.67%) in blood flow to day 21 CL. Also, there was weak positive correlation between LBF
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and turbulence to blood flow in day 12 CL (r=0.19, P < 0.01) compared to strong positive
203
correlation on day 21 CL (r= 0.84, P < 0.01). Plasma P4 concentrations were significantly higher
204
(P < 0.05) on day 21 compared to day 12 (Table 1).
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The different types of complications in pregnancy observed are described below. Intrauterine
206
growth retardation (IUGR) was observed in two cows. The fetus on day 60 had a CRL of 46 mm
207
assuming an “L” shape in one cow (Fig 2 c ) and 40 mm assuming a “C” shape in another cow
208
(Fig 2 d). Ossification of the spinal column was seen in both the fetuses but ossification of the
209
ribs was not evident (Fig 2 c). Amniotic membrane was evident with clear fluids. Fetal
210
movements were sluggish. Late embryonic death (LED) (n=2) led to loss of embryo at 25 and 28
211
days in two cows. Two cows that were declared pregnant had numerous echogenic debris
212
floating within the amniotic cavity (Fig 2 e), increased endometrial thickening and placentomes
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with abnormal contour (Fig 2 f) on day 60 compared to cows with normal pregnancy (Fig 1d).
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However, both the animals sustained the fetus to term and were found to be positive for Johne’s
215
disease.
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3.2 Characteristics of blood flow of multiple dominant follicles and CL in cows (n=3).
217
Although there were multiple follicles, the follicle with greater V and FBF (Fig 3 a) ovulated
218
within 24h and the co-dominant follicle with lower V and FBF (Fig 3 b) sustained beyond 24 h in
219
two cows (n=2) (Table 2). A corpus hemorrhagicum was observed in place of the ovulated follicle
220
24h following AI (n=2) and a single CL on day 12. However in one cow, the multiple dominant
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follicles had no blood supply (Fig 3 c) with remnants of previous CL (Fig 3 d). A highly
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vascularized follicle, good uterine tone and clear cervio-vaginal mucous was observed on day 12.
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However, insemination was not performed. All the cows had a CL on day 21 (Table 2).
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None of the cows with multiple dominant follicles had multiple ovulations as there was only one
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fetus on day 60 (n=2).
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4. Discussion
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The comparison between pregnant, non-pregnant and complicated pregnancy in cows was done
229
on the basis of period (Day of AI, Day 12 and day 21), characteristics of follicle (SA, V, FBF)
230
and CL (SA, V, LBF), presence of turbulence in blood flow to CL (day 12 vs 21), and plasma
231
concentration of P4 (day 12 vs day 21) (Table 1). It was observed that there was no significant
232
difference (P > 0.05) in BCS (Range: 3.8 ± 0.17 to 3.9 ± 0.05) between cows among groups
233
which indicates that all the cows in this study had received adequate nutrition and the reason for
234
non-pregnancy/complicated pregnancy could lie in the characteristics of the follicle and CL.
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Doppler ultrasonography was successful in evaluating functional capacity of follicle and CL to
236
sustain pregnancy based on blood flow characteristics.
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Three different types of complications were observed throughout the study. Firstly, IUGR is a
238
well described and extensively studied phenomenon in humans, however its implications in
239
bovines have not been studied extensively. Previous reports on normal fetal characteristics have
240
reported a mean CRL, TD and SkD of 60 to 70mm, 17 to 22mm, and 16 to 18mm respectively
241
[17] which were comparable to the results in our study. The initial centre’s of ossification at two
242
months are skull and vertebrae [18] which was also observed in cows with normal pregnancy.
243
IUGR can be defined as impaired growth and development of the mammalian embryo/fetus or its
244
organs during pregnancy [19] which is best determined by measuring CRL [20]. At
245
approximately day 45 of gestation, the fetus loses its rudimentary embryonic shape, and its face,
246
neck, limbs, and tail lengthen and become more defined [21]. A C-shaped fetus is generally
247
found till 35 days and an “L” shaped fetus is found between 35 to 55 days [22]. Due to reduced
248
amount of fluids, minimal ossification centre, low CRL and presence of typical embryonic shape
249
(Fig 2c,d), both the cases were suggestive of IUGR.
250
Secondly, LED has been defined as the death of the embryo between days 25 and 45 of gestation
251
[23] which was observed in two cows. We postulate that suprabasal P4 concentration caused a
252
delay in ovulation due to persistence of the regressing CL. Owing to delay in ovulation, follicular
253
evacuation was prolonged thereby leading to invasion of septae into the antrum and reduced
254
vascularisation (Fig 2 b) compared to day of AI (Fig 2 a). Such findings were reported in mares
255
[24], but not in bovines. There was a marked delay in the rise in P4 concentrations after ovulation
256
(P4 concentration of 1.45 ng/mL on day 12) which could have led to reduced production of IFN-τ
257
that has a major anti-luteolytic action. This led to luteolysis, LED and return to estrus. Similar
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findings with respect to delay in ovulation and late rise in P4 leading to embryonic death has
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been reported [25,26]. Thirdly, the effect of mycobacterium paratuberculosis sp, causative agent
260
of Johne’s disease, on the success of carrying a fetus to term requires further study.
261
On the day of AI, there was little variation (P > 0.05) in SA, V and mean diameter between
262
follicles of pregnant (PF), complicated (CPF) or non-pregnant (NPF) cows. There existed
263
statistically significant variation between FBF to PF and NPF (P = 0.01) and between PF and
264
CPF (P = 0.05). FBF proved to be a better parameter compared to diameter to assess the fate of
265
the follicle and its impact on conception rate as there was no significant variation in follicular
266
diameter among the three groups (Fig 4 a,d,g). These results agree with previous studies which
267
suggested a positive relationship between the extent of blood flow to the preovulatory follicle
268
and successful establishment of pregnancy in cattle [6]. This also disproved the previous notion
269
that a greater follicular diameter indicates a good quality follicle. Our study indicated that it is
270
important to focus on FBF irrespective of follicular diameter. In the present study cows with
271
highly (> 550 pixel2), moderately (250 to 550 pixel2) or poorly (< 250 pixel2) vascularized
272
follicles led to normal, complicated or non- pregnancy. Also, when the follicular blood flow was
273
< 250 pixel2 at the time of AI, only 25 % of cows became pregnant or had complicated
274
pregnancy, whereas a greater percentage were non-pregnant (50%.) It has been suggested that
275
the control of oocyte maturation and embryo viability resides in the follicle rather than in the
276
oocyte [27]. Poorly vascularized follicles produce very low quantities of E and accumulate
277
greater quantities of P4 both in follicular fluid and plasma [28]. This rise in P4 has a negative
278
effect on LH pulse frequency [29], thereby delaying ovulation and affecting oocyte maturation
279
which eventually leads to return to estrus. However, moderately vascularized follicles could
280
trigger significant production of E (but lower than highly vascularized follicles) which might
281
regulate LH pulses. During ovulation, the invasion of thecal cells and blood vessels into the
282
granulosa cells is necessary for production of early pregnancy factor (EPF) and transformation
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into a good quality CL along with an early switch in P4 production [26]. Therefore, moderately
284
vascularized follicles could produce less EPF leading to complications in pregnancy compared to
285
highly vascularized follicles (> 550 pixel2) which may be the limiting step which decides
286
whether a pregnancy should proceed towards normalcy or complication.
287
On day 12, there was no significant variation (P > 0.05) between mean diameter, SA, V and LBF
288
to CL or plasma P4 concentration among cows that remained pregnant (PCL), non-pregnant
289
(NPCL) or that had a complicated pregnancy (CPCL). There was weak correlation between LBF
290
and turbulence in blood flow in both PCL (r= 0.12, P < 0.0001) and CPCL (r=0.19, P < 0.01).
291
None of the parameters assessed on day 12 were useful in differentiating among the three groups
292
(Fig 4 b,e,h). However, day 21 CL was a better indicator as there were significant differences on
293
comparison of parameters between the three groups. Except for SA between PCL and CPCL
294
(P>0.05), there was statistically significant variation between SA, V and P4 concentration
295
between PCL, CPCL and NPCL (Table 1). LBF alone was not beneficial in differentiating
296
among the three groups (P > 0.05), but interesting results were obtained on evaluation of LBF
297
along with turbulence to blood flow in day 21 CL (Fig 4 c,f,i). There was weak correlation
298
between LBF to PCL and turbulence to blood flow (r= 0.09, P < 0.0001), but there was a strong
299
positive correlation between LBF to CPCL and turbulence to blood flow (r= 0.84, P < 0.01).
300
Turbulence to day 21 CL was observed in all NPCL (Table 1). It was also observed that there
301
was significant increase in LBF (1.7 fold) and V (P < 0.05) from day 12 to 21 in pregnant cows,
302
but there was no significant increase in LBF and V (P > 0.05) from day 12 to 21 in cows with
303
complicated pregnancy. A 2.6 fold increase in LBF and 3.7 fold decrease in V in NPCL with
304
significant turbulence was observed on day 21 when compared to day 12 which indicated the
305
start of luteolysis. Thus assessment of turbulence and increase in LBF from 12 to 21 days post AI
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can give a fair indication of the fate of a CL. Assessment of turbulence and LBF can be used to
307
predict luteolysis which can save time and increase profitability in a dairy farm.
308
A delayed rise in P4 during early pregnancy (as seen on comparison of pregnant cows and cows
309
with complicated pregnancy in Table 1) suggests luteal inadequacy and it has been reported that
310
cows that underwent an earlier rise in P4 had embryos that were further developed and produced
311
more of the antiluteolytic protein, interferon –tau (IFN-τ) by day 16 than cows that had a delayed
312
rise in concentrations of P4 [25]. IFN-τ along with P4 secreted by CL exerts their antiluteolytic
313
effect by modifying oxytocin receptors inhibiting the synthesis from arachidonic acid and
314
subsequent release of PGF2α [30]. The maximum secretion of IFN-τ occurs between days 16-19
315
of gestation [31]. The results from previous studies indicate that low P4 concentrations during an
316
estrus cycle have a delayed stimulatory effect on uterine responsiveness to oxytocin during the
317
late luteal phase of the subsequent cycle [32]. The resulting increase in PGF2α secretion may
318
interfere with luteal maintenance during the early stages of pregnancy [32]. This could suggest
319
that the suboptimal P4 in cows with complicated pregnancy (day 12; Table 1) was insufficient in
320
preventing the release of PGF2α, thereby affecting the growth and survival of the fetus.
321
Turbulence in blood flow, though subjective, was minimum in PCL compared to NPCL (P <
322
0.01) and CPCL (P < 0.05). Thus, a minimal release of PGF2α might be responsible for the
323
increase in turbulence in day 21 CPCL in cows which may or may not cause death of the fetus,
324
but the success in carrying it to term may be hampered and requires further study.
325
These findings suggested that LBF, turbulence to CL and plasma P4 concentration on day 21
326
were valuable indicators reflecting the functional capacity of the CL between different groups.
327
These results agree with previous studies which indicate that LBF is a reliable indicator of luteal
328
status [33]. However, our study adds turbulence to blood flow as a new parameter along with
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LBF. Plasma P4 concentration remains the gold standard for evaluation of luteal function [34].
330
But P4-based diagnosis is time-consuming, hence difficult to obtain results immediately after
331
sampling. Therefore colour Doppler ultrasonography can be a useful cow-side tool for immediate
332
assessment of ovarian structures and prediction of functional status.
333
CL at the time of AI was observed in all groups. However, LBF determined the fate of the CL. A
334
regressing CL present on the day of AI (Fig 5a) had increased LBF and turbulence 24h following
335
AI (Fig 5b) suggesting that it was undergoing complete luteolysis and the functional capacity of
336
the CL in producing P4 was very low leading to a normal pregnancy. However, a persistent CL
337
on the day of AI (Fig 5c) had decreased LBF and absence of turbulence 24h following AI (Fig
338
5d). The prolonged life span of the CL caused delay in ovulation of follicle due to the negative
339
impact of P4 produced by the CL on LH pulses attributing to LED. The negative effects of P4
340
have also been reported in several other studies [35,36]. These findings suggest that assessment
341
of vascularization to regressing CL revealed different patterns of blood flow which can be
342
effective in predicting the pattern of regression and effect on pregnancy.
343
Although two cows had multiple dominant follicles, multiple ovulations were not observed
344
because there was a difference in vascularization between the multiple dominant follicles and
345
only the highly vascularized follicle ovulated whereas the co-dominant follicle persisted and
346
regressed at a later stage. Thus it can be concluded that Doppler ultrasonography can be a useful
347
aid in detection of multiple ovulation which can lead to twins.
348
5. Conclusion
349
Assessment of vascularization to follicle (FBF) on the day of AI and CL (LBF and turbulence to
350
blood flow) during early pregnancy using Doppler ultrasonography proved to be a successful
351
predictor of fertility. Cows with a highly vascularized follicle (> 550 pixel2) underwent a normal
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pregnancy, whereas those that had moderately (250 to 550 pixel2) and poorly (< 250 pixel2)
353
vascularized follicle experienced complicated pregnancy or remained non-pregnant, respectively.
354
On day 21, LBF alone was not beneficial in differentiating among the three groups, but
355
assessment of LBF along with turbulence to blood flow in day 21 CL proved highly valuable due
356
to an increased turbulence in CPCL (66.67%) compared to PCL (16.67%). Assessment of
357
turbulence and LBF can also be used to predict luteolysis in CL.
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Acknowledgements
360
The authors thank the faculty and staff of the Department of Veterinary Gynaecology and
361
Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) for their
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help and advice. We show our sincere gratitude to the working staff and faculty at the Dairy
363
Farm Complex and dairy farms for their continued support.
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365 References
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366 [1] Breuel KF, Lewis PE, Schrick FN, Lishman AW, Inskeep EK, Butcher RL. Factors affecting 367 fertility in the postpartum cow: Role of oocyte and follicle in conception rate. Biol Reprod 368 1993; 48: 655-61.
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369 [2] Brannstrom M, Zackrisson U, Hagstrom HG, Josefsson B, Hellberg P, Granberg S, Collins 370 WP, Bourne T. 1998. Preovulatory changes of blood flow in different regions of the human 371 follicle. Fertil Steril 69: 435-42. 372 [3] Ginther OJ. Arteries and Hemodynamics. In: Ginther OJ. Ultrasonic Imaging and Animal 373 Reproduction: Color-Doppler Ultrasonography, Wisconsin: Equiservices Publishing, Cross 374 Plains; 2007, p. 7-24. 375 [4] Coulam CB, Goodman C, Rinehart JS. Colour Doppler indices of follicular blood flow as 376 predictors of pregnancy after in-vitro fertilization and embryo transfer. Hum Reprod 1999; 14: 377 1979–82.
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378 [5] Huey S, Abuhamad A, Barroso G, Hsu MI, Kolm P, Mayer J, Oehninger S. Perifollicular blood 379 flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental 380 competence in in vitro fertilization. Fertil Steril 1999; 72: 707–12.
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381 [6] Siddiqui MAR, Almamun M, Ginther OJ. Blood flow in the wall of the preovulatory follicle 382 and its relationship to pregnancy establishment in heifers. Anim Reprod Sci 2009; 113: 287– 383 92. 384 [7] Kastelic JP, Bergfelt DR, Ginther OJ. Relationship between ultrasonic assessment of the corpus 385 luteum and plasma progesterone concentration in heifers. Theriogenology 1990a; 33: 1269386 78.
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387 [8] Bergfelt DR, Ginther OJ. Ovarian and embryo dynamics in horses versus ponies. J Equine Vet 388 Sci 1996; 16: 66-72.
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389 [9] Kawakami S, Shida T, Mutoh M, Kohmoto H, Onhchi T. Relation between luteal regression 390 and so-called counter current mechanisms in the cow: Verification from PGF2α 391 concentrations in arterial, uterine venous and jugular venous blood following PGF2α loading. 392 J Reprod Dev 1995; 41: 219–23. 393 [10] Acosta T J, Yoshizawa N, Ohtani M, Miyamoto A. Local changes in blood flow within the 394 early and midcycle corpus luteum after prostaglandin F(2 alpha) injection in the cow. Biol 395 Reprod 2002; 66: 651–8.
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396 [11] Carrière PD, Gnemmi G, Descôteaux L, Matsui M, Miyamoto A, Colloton J. Bovine ovary. 397 In: Des Côteaux L, Colloton J and Gnemmi G. Practical atlas of ruminant and camelid 398 reproductive ultrasonography, United Kingdom: Wiley-Blackwell publishers; 2010, p. 35-60. 399 [12] Ginther OJ, Utt MD. Doppler Ultrasound in Equine Reproduction: Principles, Techniques, 400 and Potential. J Equine Vet Sci 2004; 24: 516-26.
Imaging
#4.
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401 [13]Kisslo JA, Adams DB. Doppler Color Flow 402 http://cardioland.org/Echo/doppler04.pdf [accessed on 05.02.17].
403 [14] Pierson RA, Ginther OJ. Ultrasonography of the bovine ovary. Theriogenology 1984; 21: 404 495–504.
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405 [15] Ghuman SPS, Dadarwal D, Honparkhe M, Singh J, Dhaliwal GS. Production of polyclonal 406 antiserum against progesterone for radioi-mmunoassay. Indian Vet J 2009; 86: 909-11. 407 [16] SPSS 16.0, 2007: Command Syntax Reference. SPSS Inc©, South Wacker Drive, Chicago. 408 [17] Kahn W. Sonographic fetometry in the bovine . Theriogenology 1989; 31: 1105-21. 409 [18] Gjesdal F. Age determination of bovine foetuses. Acta Vet Scand 1969; 10: 197-218. 410 [19] Wu G, Bazer FW, Wallace JM, Spencer TE. Intrauterine growth retardation: Implications for 411 the animal species. J Anim Sci 2006; 84: 2316-37. 412 [20] Mandruzzato G. Intrauterine growth restriction (IUGR): Guidelines for definition, recognition 413 and management. Arch Perin Med 2008; 14: 7-8. 17
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414 [21] Descôteaux L, Colloton J, Gayrard V, Picard-Hagen N. Bovine pregnancy, In: Des Côteaux L, 415 Colloton J and Gnemmi G. Practical atlas of ruminant and camelid reproductive 416 ultrasonography, United Kingdom: Wiley-Blackwell publishers; 2010, p. 81-99.
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417 [22] Kolour AK, Batavani RA, Ardabili FF. Preliminary observations on the effect of parity on 418 first day ultrasonic detection of embryo and its organs in bovine. J Vet Med A Physiol Pathol 419 Clin Med 2005; 52: 74-7. 420 [23] Committee on Bovine Reproductive Nomenclature. Recommendations for standardising 421 bovine reproductive terms. Cornell Veterinary 1972; 216-37.
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422 [24] Ginther OJ, Gastal EL, Gastal MO. Spatial Relationships between Serrated Granulosa and 423 Vascularity of the Preovulatory Follicle and Developing Corpus Luteum. J Equine Vet Sci 424 2007; 27: 20-7.
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425 [25] Mann GE, Lamming GE. Relationship between maternal endocrine environment, early 426 embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 2001; 427 121: 175-80. 428 [26] Hafez ESE, Hafez B. Folliculogenesis, egg maturation and ovulation. In: Hafez ESE, Hafez 429 B. Reproduction in Farm Animals, Pennsylvania: Lipincott Williams and Wilkins; 2000, p.79. 430 [27] Moor RM, Dai Y, Lee C, Fulka JJr. Oocyte maturation and embryonic failure. Human 431 Reproduction 1998; 4: 223–36 (Update).
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432 [28] Varughese EE. Assessment of the vascularization of ovarian structures and their correlation 433 with fertility in dairy animals, M.V.Sc Thesis, Guru Angad Dev Veterinary and Animal 434 Sciences University, Ludhiana, Punjab, India. 2012. 435 [29] Adams GP, Matteri RL, Ginther OJ. Effect of progesterone on ovarian follicles, emergence of 436 follicular waves and circulating follicle-stimulating hormone in heifers. J Reprod Fertil 1992; 437 95: 627–40.
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438 [30] Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferon-τ production 439 and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999; 54: 317-28 440 (Supple).
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441 [31] Bartol FF, Roberts RM, Bazer FW. Characetrization of proteins produced by peri-attachment 442 bovine conceptuses. Biol Rerod 1985; 32: 681-93. 443 [32] Wolfenson D. Factors associated with low progesterone concentrations and their relation to 444 low fertility of lactating dairy cows. Israel J Vet Med 2006; 61. 445 [33] Herzog K, Brockhan-Ludemann M, Kaske M, Beindorff N, Paul V, Niemann H, Bollwein H. 446 Luteal blood flow is a more appropriate indicator for luteal functionduring the bovine estrous 447 cycle than luteal size. Theriogenology 2010; 73: 691–7. 448 [34] Kastelic JP, Pierson RA, Ginther OJ. Ultrasonic morphology of corpora lutea and central 449 luteal cavities during the estrous cycle and early pregnancy in heifers. Theriogenology 1990b; 450 34: 487–98
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451 [35] Gustafsson H, Larsson K, Kindahl H, Madej A. Sequential endocrine changes and behavious 452 during estrus and metestrus in repeat breeder and virgin heifers. Anim Reprod Sci 1986; 19: 453 261-73.
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454 [36] Albihn A, Gustafsson H, Hurst M, Rodriguez-Martinez H. Embryonic ability to prolong the 455 interestrus interval in virgin and repeat breeding heifers. Anim Reprod Sci 1991; 26: 193-210.
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Table 1
Characteristics of CL on day 12
SA (mm2)
V (mm3)
FBF (pixel2)
Mean diameter (mm)
SA (mm2)
V (mm3)
LBF (pixel2)
15.98 ± 1.73
164.92 ± 17.67
2011.23 ± 306.88
609.33 ± 89.94a*a
19.06 ± 1.22
252.07 ± 14.54
3429.48 ± 296.37g
1868.67 ± 121.83g
14.31 ± 2.23
117.52 ± 18.76
1352.38 ± 298.94
199.40 ± 68.26b
20.35 ± 2.55
Complicated Pregnancy (n=6)
14.25 ± 1.35
134.11 ± 19.61
1410.87 ± 240.72
286.40 ± 83.03c
18.82 ± 1.99
256.67 ± 34.56
228.13 ± 30.11
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NonPregnant (n=5)
Characteristics of CL on day 21
P4 (ng/ mL)
Mean diameter (mm)
SA (mm2)
V (mm3)
LBF (pixel2)
P4 (ng/ mL)
T (% of cases )
2.19 ± 0.11
21.68 ± 2.14
304.42 ± 17.94d
4947.95 ± 286.43a*
3124.50 ± 243.34f
3.36 ± 0.23a*
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Mean diameter (mm)
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Characteristics of follicle
df
bc
d
3464.43 ± 819.29
1265.00 ± 174.46
2.30 ± 0.24
12.37 ± 0.58
109.30 ± 7.23e
946.58 ± 124.16e
3268.50 ± 772.50
0.79 ± 0.21be
100 a
3191.47 ± 485.17
1961.17 ± 418.04g
1.83 ± 0.21
19.80 ± 1.62
260.20 ± 14.80d
3722.37 ± 284.16cd
2771.83 ± 348.45f
2.51 ± 0.20ac
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Comparison between pregnant, non-pregnant and complicated pregnancy in cows on the basis of characteristics of follicle, CL, turbulence to CL and plasma progesterone (P4) concentration (excluding cows with multiple dominant follicles).
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a represents significantly (P ≤ 0.01) higher than b within columns between similar variables. a* represents significantly (P ≤ 0.05) higher than c within columns between similar variables. d represents significantly (P ≤ 0.0001) higher than e within columns between similar variables. f represents significantly (P ≤ 0.05) higher than g within rows between similar variables.
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Table 2
Animal No.
Characteristics of follicles on day of AI
FBF 24 h of AI
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Morphological evaluation and characteristics of blood flow of multiple dominant follicles on the day of artificial insemination (AI), 24h following AI and CL on day 12 and 21 following AI in cows.
Characteristics of CL on day 12
2*.
3^.
SA (mm2)
176.71
2506.48
821
-
84.95
837.20
243
400
211.24
2489.79
515
-
141.03
1727.94
64
250
88.25
781.56
0
0
88.25
646.41
0
0
LBF (pixel2)
V (mm3)
Characteristics of cavity
P4 (ng/ mL)
SA (mm2)
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FBF (pixel2)
V (mm3)
LBF (pixel2)
V (mm3)
Type
-
-
1.70
350.67
4955.64
2822
Presence of turbulence
Characteristics of cavity
P4 (ng/ mL)
V (mm3)
Type
-
-
-
3.0
263.02
3765.67
1987
593.96
9513.48
2146
1363.32
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2.65
650.78
9899.65
3022.12
-
874.32
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3.3
-
-
-
-
-
0.33
543.25
9586.5
2610
+
1062.34
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1.7
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1*.
V (mm3)
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SA (mm2)
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(pixel2)
Characteristics of CL on day 21
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*1st follicle ovulated and the 2nd follicle was present ^Cow came in heat on day 12 with clear and thick estrous discharge and strong uterine tone, but insemination was not performed.
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502
Figure legends
503
Fig. 1. Pregnancy diagnosis on day 60 in cows with normal pregnancy (a) Ossification of ribs and spinal column (yellow arrows), (b) Ossification centres around maxillary and mandibular bones, (c) An intact amniotic membrane overlying the fetus (red arrow), (d) Placentomes were seen as small eruptions and offset indicates the blood flow to placentomes, (e) LBF on day 60 indicating copious blood supply with mild turbulence (yellow arrow) suggestive of greater velocity in blood flow to supply the pregnant CL, (f) An incompletely luteinized CL of day 21 replaced by a hyperechogenic scar (red arrow) on day 60, (g) Persistence of central cavity in day 60 CL with development of a hyperechogenic ring (orange arrow) around the cavity,
513 514 515 516 517 518 519 520 521
Fig. 2. Blood flow to follicle on day of artificial insemination (AI) and pregnancy diagnosis on day 60 in cows with complicated pregnancy (a) Blood flow to follicle on day of AI (530 pixel2), (b) Prolonged sustenance of the follicle with reduction in blood flow (133 pixel2) and initiation of formation of septae (white arrow) within the follicular antrum, 24 hrs following AI, (c) L-shaped fetus with a crown-rump length (CRL) of 46 mm and ossification of spinal column (black arrow) with no evidence of ossification of ribs, (d) Doppler on C-shaped fetus with a CRL of 40 mm showing blood supply to fetus. (e) Echogenic debris within amniotic cavity, (f) Increased endometrial thickening (red arrow) and placentomes with abnormal contour.
522 523 524 525 526
Fig. 3. Characteristics of multiple dominant follicle’s. (a) Follicle with greater volume and vascularization on the day of AI (515 pixel2) (b) Co-dominant follicle with lower volume and vascularization on the day of AI (64 pixel2), (c) Multiple follicle’s in Cow 3 with absence of blood supply on the day of AI, (d) Remnants of CL were observed on the day of AI which is evident from the increased vascularization.
532 533 534 535 536 537
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Fig. 4. Assessment of vascularization to follicle and CL in three groups. Pregnant cow - (a) Follicular blood flow (FBF) on the day of AI (934 pixel2), (b) Day 12 CL (1758 pixel2), (c) Day 21 CL (3948 pixel2). Note the increase in size of blood vessels (orange arrow) on day 21 compared to day 12 and absence of turbulence in blood flow on both day 12 and day 21 CL.
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527
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504 505 506 507 508 509 510 511 512
Complicated pregnancy (Intrauterine growth retardation) (d) FBF on the day of AI (340 pixel2), (e) Day 12 CL (2716 pixel2), (f) Day 21 CL (3209 pixel2). Turbulence was present in both day 12 and 21 CL (yellow arrow). There was no considerable increase in size of the CL or blood vessels in day 21 CL compared to day 12 CL. Non-pregnant cow– (g) FBF on the day of AI (139 pixel2), (h) Day 12 CL with an eccentric cavity and absence of turbulence (1362 pixel2), (i) Day 21 CL (bottom) with
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a significant increase in vascularization (4041 pixel2), turbulence (white arrow), reduction in size indicative of luteolysis and presence of follicle (top).
540 541 542 543 544 545 546
Fig. 5. Luteolysis and fate of corpus luteum (CL) assessed by vascularization and turbulence in luteal blood flow (LBF). (a) CL on the day of AI with LBF (5788 pixel2) and minimal turbulence, (b) CL with increased LBF (6509 pixel2) and greater turbulence (yellow arrow) 24 hrs following AI which suggests that CL might undergo complete luteolysis, (c) CL on day of AI with good LBF (6364 pixel2) and moderate turbulence (yellow arrow), (d) CL 24 hrs following AI with decreased LBF (3959 pixel2) and absence of turbulence which suggests a persistent CL.
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Highlights Predict pregnancy rate based on vascularization to follicle and corpus luteum (CL).
•
Cows with a highly vascularized follicle (> 550 pixel2) underwent a normal pregnancy.
•
Moderately (250 to 550 pixel2) and poorly (< 250 pixel2) vascularized follicle
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experienced complicated pregnancy or remained non-pregnant, respectively. •
Assessment of luteal blood flow along with turbulence to blood flow in day 21 CL
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pregnant cows (16.67%).
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Increased turbulence in CL of complicated pregnant cows (66.67%) compared to CL of
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proved highly valuable.