Reprod Dom Anim 49, 985–988 (2014); doi: 10.1111/rda.12418 ISSN 0936–6768
Accurate Ultrasonographic Prediction of Progesterone Concentrations Greater than 1 ng/ml in Holstein lactating dairy cows K Kaneko1 and N Takagi2 1 Department of Veterinary Obstetrics and Gynecology, Azabu University, Chuo-ku, Sagamihara, Kanagawa, Japan; 2Komagatake Ohnuma Veterinary Clinic, Kayabe-gun, Hokkaido, Japan
Contents To develop an ultrasonographic assay for determining plasma progesterone concentration (P4) as < 1 ng/ml or ≥ 1 ng/ml, the corpus luteum (CL) area and P4 were measured in 1094 multiparous Holstein cows. The area-measuring function and frozen images were used to outline and measure CL imaged via ultrasonography, and CL area was estimated as a polygon of a continuation straight line. A significant correlation was found between CL area and P4 (p < 0.001), and this analysis resulted in the following correlation equation: y = 0.35 + 1.02x (r = 0.81). According to the correlation equation, a CL area of 1.3 cm2 indicated a P4 of 1 ng/ml. Based on this relationship, each animal was categorized into one of six groups, groups differed based on CL area, and the area ranges were as follows: < 1.3 cm2 (Group A), 1.3–2.2 cm2 (Group B), 2.3– 3.2 cm2 (Group C), 3.3–4.2 cm2 (Group D), 4.3–5.2 cm2 (Group E) and > 5.2 cm2 (Group F). For each group, the proportion of cows whose P4 was 1 ng/ml or more was 1.5% in Group A, 83.3% in Group B, 76.6% in Group C, 96.6% in Group D, 99.2% in Group E and 100% in Group F. There was a significant difference between Group A and the other five groups, and between Groups B or C and Groups D, E or F (p < 0.005). These results indicate that a functional CL does not exist when the CL area is less than 1.3 cm2 and that it exists when the CL area is 3.3 cm2 or more.
Introduction In the dairy industry, it is important to monitor postpartum ovarian activity (Aldrich et al. 1995; Aungier et al. 2012; Nebel 1988; Petersson et al. 2008; Rekwot et al. 2004; Samar€ utel et al. 2008) and evaluate the response to endocrine therapy (Nebel 1988; Tallam et al. 2001; Tenhagen et al. 2000) as the reproductive management. For these purposes, progesterone assays involving milk or blood samples have been used (Claycomb and Delwiche 1998; Østergaard et al. 2005). However, the accurate progesterone assay demands specific equipment and time. The corpus luteum (CL) is the endocrine organ responsible for progesterone production, and it is more important to qualify reliably the CL as functional or not functional as soon as possible than to know the precise progesterone profiles for several clinical purposes. To successfully integrate into a reproductive management protocol, any test needs to be (i) sensitive, (ii) specific (i.e. identify correctly functional from non-functional CL), (iii) inexpensive, (iv) simple to conduct under field conditions and (v) able to determine CL status at the time the test is performed. Because P4 > 1 ng/ml is considered to indicate the presence of functional luteal tissue (Rajamahendran et al. 1993; Silva et al. 2007a), the most important thing to know is whether the P4 is < 1 ng/ml © 2014 Blackwell Verlag GmbH
or ≥ 1 ng/ml. Ultrasonography is used to examine the ovarian structures in cattle. The objective of this study was to make a dividing line that separates P4 into < 1 ng/ml or ≥ 1 ng/ml by ultrasonography on the field, which will be able to use for the reproductive management.
Materials and Methods Animals This study was conducted with 1094 multiparous Holstein cows from nine dairy farms. The cows were housed in a barn stall, fed a total mixed ration and had free access to water. The age of cows was 3.9 0.8 (Mean SEM), and their BCS was in the range of 2.5– 4.0. Each cow was examined 30–120 days post-partum and had received a regular rectal examination every 2 weeks as part of a reproductive management protocol. Each cow that was confirmed to be pregnant or to have abnormalities in their ovaries or uterus clinically (cystic follicles, persistent ovarian follicles, endometritis and pyometra) was excluded from data analysis. Ultrasound scanning A real-time ultrasonograph (Model HS-2100V; Honda Electronics Co., Ltd, Aichi, Japan) equipped with a 10MHz transrectal linear transducer was used to measure each CL area. The area-measuring function of the ultrasonograph and frozen image were used to trace the CL outline and take each measurement. The crosssectional area of the ultrasonic section of CL was estimated as a polygon of a continued straight line. This method considers a curve to be a continuation of a short straight line. After the line is processed as pixel (dot), it is converted into mm, and then, the area is computed. When a central cavity was present in a CL, the area of the cavity was subtracted from the total area (Kaneko et al. 2004). Blood sampling A vacuum-type heparinized tube was used to collect each blood sample from the tail vein. Each sample was collected when the corresponding CL was observed. Plasma was separated by centrifugation (2000 9 g for 10 min) and stored at 80°C until the determination of P4. Hormone analysis P4 was measured using a commercial radioimmunoassay kit for progesterone (Diagnostic Products, Los Angeles,
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CA, USA), which has been previously validated for cattle (Peter and Bous 1987). Intra- and interassay coefficients of variation were 8.8% and 9.7%, respectively; the assay average sensitivity was 0.02 ng/ml. Data analysis Linear regression analysis was used to determine the correlation between CL area and P4. A strong correlation was found between CL area and P4 (p < 0.001, R = 0.81), and this analysis resulted in the following correlation equation: y = 0.35 + 1.02x (y, P4; x, the CL area) (Fig. 1). According to the correlation equation, a CL area of 1.3 cm2 should indicate a P4 of 1 ng/ml. To assess the relationship between CL area and the P4 > 1 ng/ml, each pair of CL area and P4 measurements was assigned to one of six groups, where the groups were based on CL area. Group A comprised areas < 1.3 cm2, Group B areas between 1.3 and 2.2 cm2, Group C areas between 2.3 and 3.2 cm2, Group D areas between 3.3 and 4.2 cm2, Group E areas between 4.3 and 5.2 cm2, and Group F areas greater than 5.2 cm2. Logistic regression method was used to assess the significance of the rate whose P4 was 1 ng/ml or more (high P4 rate) among groups. p values < 0.01 were considered statistically significant.
Results The high P4 rate according to the CL area was 1.5% (5/329) for Group A, 83.3% (165/198) for Group B, 76.6% (134/175) for Group C, 96.6% (172/178) for Group D, 99.2% (119/120) for Group E and 100% (94/94) for Group F (Fig. 2). The high P4 rate in Group A was significantly lower than that in Group B, C, D, E and F (p < 0.005). Also, there was a significant difference between Group B or C and Group D or E or F (p < 0.005). The high P4 rate of Group B + Group C was 80.2% (299/373) and that of Group D + Group E + Group F was 98.2% (385/392).
Discussion Although a relationship between the CL size and the P4 was reported, CL size was estimated by computing the area or the volume using CL height and CL width, and 14 12 10 8 6 4 2 0 –2
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Fig. 1. Relationship between progesterone concentration and corpus luteum area. Correlation coefficient: r = 0.81, p < 0.001, P4, progesterone; CL, corpus luteum
Fig. 2. Rates of P4 ≥ 1 ng/ml in each group. Group A: CL area less than 1.3 cm2, Group B: CL area between 1.3 and 2.2 cm2, Group C: CL area between 2.3 and 3.2 cm2, Group D: CL area between 3.3 and 4.2 cm2, Group E: CL area between 4.3 and 5.2 cm2, Group F: CL area greater than 5.3 cm2. There is a significant difference between Group A and Group B, C, D, E, F and between Group B or C and Group D or E or F (p < 0.005), P4, progesterone; CL, corpus luteum
the reported correlation coefficient was 0.31–0.77 (Kastelic et al. 1990a; Son et al. 1995; Sartori et al. 2002; L€ uttgenau et al. 2011). In this study, the area-measuring function of the ultrasonograph was used to measure the CL area as a polygon of a continued straight line. This method considers a curve a continuation of a short straight line, and computes the area accordingly. The resulting correlation coefficient between the CL area and P4 (0.81) is stronger than the previously reported correlations (Kastelic et al. 1990a; Son et al. 1995; Sartori et al. 2002; L€ uttgenau et al. 2011). For efficient reproductive management of dairy cows, it is important to know whether P4 is above 1 ng/ml, because this P4 value is a strong indicator of a functional CL (Rajamahendran et al. 1993; Silva et al. 2007a). Based on the correlation equation determined here, a CL area of 1.3 cm2 indicated a P4 of 1 ng/ml. For CL area measurements of less than 1.3 cm2 (Group A), the high P4 rate was only 1.5%. This suggests that functional CL does not exist when CL area measurements are less than 1.3 cm2. However, for cases where CL area was 3.3 cm2 or more (Group D, E and F), the high P4 rate was 98.2%. Therefore, it should be very possible to determine that a CL is functional when CL area measurements are 3.3 cm2 or more. On the other hand, when CL area was between 1.3 and 3.2 cm2 (Group B and C), it is difficult to determine whether the functional CL exists or not. The probability of incorrectly estimating P4 as ≥ 1 ng/ ml when the CL area was less than 1.3 cm2 was 1.5%, and probability of incorrectly estimating P4 as < 1 ng/ml when the CL area is 3.3 cm2 or more was 1.8%. However, in cases where CL area was 1.3–3.2 cm2 (Group B + Group C), the P4 was incorrectly estimated as 1 ng/ml or more even if it was less than 1 ng/ml in 19.8% of cows. The CL grows quickly when it is developing; conversely, it regresses quickly when it is degenerating. In heifers, CL maintains an area of 1.3– 3.2 cm2 for about only 2 days when it is developing and again later for 2 days when it is degenerating (Kastelic et al. 1990b). Although this criterion cannot be applicable for multiparous cows, P4 fluctuates significantly during these two periods in multiparous cows (Kaneko and Kawakami 2008). Because of that, it is quite © 2014 Blackwell Verlag GmbH
Ultrasonographic Prediction of Progesterone Concentrations
possible that P4 may be misjudged on around this CL size. Assey et al. (1993) investigated the relationship between CL size and P4 in the developing and in the degeneration periods. They found that the correlation coefficient between the CL size and the P4 was only 0.26 or 0.47. Based on Assey et al. (1993) and findings from the present study, it might be possible to reduce the misjudgement of P4 by rechecking the CL area within a few days for any CL area measurement between 1.3 and 3.2 cm2. Furthermore, in the future, if a method which can distinguish developing CL from regressing CL by ultrasonography will be developed, it may be possible to reduce the misjudgement of P4. Notably, Sing et al. reported that it was possible to allocate the CL to metoestrus, mid-dioestrus and pro-oestrus by observing the enucleated ovaries by ultrasonography (Singh et al. 1997). On the other hand, ultrasonography was inaccurate for the identification of young or old CL in the ovaries in vivo (Pieterse et al. 1990), and ultrasonographic diagnosis of the growing or regressing CL was difficult (Hanzen et al. 2000). When the blood flow to the CL was observed by color Doppler, the blood supply to the developing CL increased in parallel with its growth (Acosta et al. 2003; Miyamoto et al. 2006; Matsui and Miyamoto 2009) and decreased just before the luteolysis (Acosta et al. 2002). Consequently, the combination of the measurement of CL area and the observation of the blood flow to the CL may help to reduce misjudgements. We measured the cross-sectional area of the CL as the CL size instead of cubic volume or weight in this study, and this way of measurement is thought to be one of the
References Acosta TJ, Yoshizawa N, Ohtani M, Miyamoto A, 2002: Local changes in blood flow within the early and midcycle corpus luteum after prostaglandin F2a injection in the cow. Biol Reprod 66, 651–658. Acosta TJ, Hayashi KG, Ohtani M, Miyamoto A, 2003: Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows. Reproduction 125, 759–767. Aldrich SL, Berger LL, Reiling BA, Kesler DJ, Nash TG, 1995: Parturition and periparturient reproductive and metabolic hormone concentrations in prenatally androgenized beef heifers. J Anim Sci 73, 3712–3718. Assey RJ, Purwantara B, Greve T, Hyttel P, Schmidt MH, 1993: Corpus luteum size and plasma progesterone levels in cattle after cloprostenol-induced luteolysis. Theriogenology 39, 1321–1330. Aungier SP, Roche JF, Sheehy M, Crowe MA, 2012: Effects of management and health on the use of activity monitoring for estrus detection in dairy cows. J Dairy Sci 95, 2452–2466. Claycomb RW, Delwiche MJ, 1998: Biosensor for on-line measurement of bovine progesterone during milking. Biosens Bioelectron 13, 1173–1180. Hanzen CH, Pieterse M, Scenczi O, Drost M, 2000: Relative accuracy of the identi-
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reasons for misjudgement. However, the correlation coefficient between the CL weight and the P4 was reported to be 0.80 (Mann 2009) or 0.25–0.98 (Robinson et al. 2005), and the findings from the present study were quite comparable. These show that the assessment of the CL size from the cross-sectional area of the CL by ultrasonography is a useful substitution for measuring the weight of the CL. The ultrasonographic assay described here reliably estimated a P4 cut-off of 1 ng/ ml in multiparous Holstein cows. The results in the present study showed that there is a strong positive correlation between the CL area, as measured by ultrasonography, and P4. Furthermore, we were able to accurately determine that P4 was less than 1 ng/ml when the CL area was less than 1.3 cm2 and P4 was 1 ng/ml or more when CL area was more than 3.3 cm2. However, in the case in which the CL area was 1.3–3.2 cm2, the P4 was misjudged as 1 ng/ml or more when it was actually less than 1 ng/ml in 19.8% of cows. These results indicate that a functional CL does not exist when the CL area is less than 1.3 cm2 and that it exists when the CL area is 3.3 cm2 or more. Author contributions Kaneko has designed the study, analysed the data, collected the samples and drafted this paper. Takagi has collected the samples for this paper.
Conflict of interest None of the authors have any conflict of interest to declare.
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K Kaneko and N Takagi Sartori R, Rosa GJM, Wiltbank MC, 2002: Ovarian structures and circulating steroids in heifers and lactating cows in summer and lactating and dry cows in winter. J Dairy Sci 85, 2813–2822. Silva E, Sterry RA, Fricke PM, 2007a: Assessment of a practical method for identifying anovular dairy cows synchronized for first postpartum timed artificial insemination. J Dairy Sci 90, 3255–3262. Singh J, Pierson RA, Adams GP, 1997: Ultrasound image attributes of the bovine corpus luteum: structural and functional correlates. J Reprod Fertil 109, 35–44. Son CH, Schwarzenberger F, Arbeiter K, 1995: Relationship between ultrasonographic assessment of the corpus luteum area and milk progesterone concentration during the estrous cycle in cows. Reprod Domest Anim 30, 97–100. Tallam SK, Kerbler TL, Leslie KE, Bateman K, Johnson WH, Walton JS, 2001: Reproductive performance of postpartum dairy
cows under a highly intervenient breeding program involving timed insemination and combinations of GnRH, prostaglandin F2alpha and human chorionic gonadotropin. Theriogenology 56, 91–104. Tenhagen BA, Birkelbach E, Heuwieser W, 2000: Serum progesterone levels in post-partum dairy cows after repeated application of the prostaglandin F2 alpha analogue D (+) cloprostenol sodium. J Vet Med A Physiol Pathol Clin Med 47, 213–220. Submitted: 18 Apr 2014; Accepted: 6 Aug 2014 Author’s address (for correspondence): K Kaneko, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan. E-mail: kaneko@azabu-u. ac.jp
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