Luteal Area Research Articles

107 Timing of progesterone exposure effects pregnancy rates following fixed time artificial insemination in postpartum beef cows

Abstract Advancements in reproductive technologies have improved pregnancy rates in postpartum cows; however, undesirable consequences leading to reduced fertility still occur. Thus, the objectives were to determine if presence of a corpus luteum (CL) at the start of estrous synchronization (AIM1) or if the length of proestrus (interval from progesterone removal to a GnRH-induced LH surge; AIM2) alters fixed timed artificial insemination (FTAI) pregnancy rates. Cycling postpartum cows (n = 285) at two locations were synchronized using the CO-Synch+CIDR® FTAI protocol. Ultrasonographic measurements of luteal area were recorded at CIDR insertion. To determine the impact of proestrus length, cows were randomly assigned to either undergo CIDR removal at the time of PGF2a injection (long proestrus, n = 145), or CIDR removal 24 hours post PGF2a injection (short proestrus, n = 140). Blood samples were collected at CIDR insertion and at insemination for progesterone (P4) analysis. Pregnancy status to FTAI was assessed using ultrasonographic diagnosis at 30 d post insemination. Data were analyzed using a MIXED procedure of SAS and examined for effects of length of proestrus, P4 concentration, luteal area, sire, location, and age. Statistical significance was determined at P0.05) pregnancy rates. Pregnancy rates were greater in cows with >1 ng/mL P4 compared to cows with 0.05). Cows > 4 y old had higher AI pregnancy rates compared to 2–3 y cows (70.2±7.4 vs. 51±5.8%, respectively; P > 0.05). Based on these data, incorporating a presynchronization program to ensure the presence of a functional CL at the start of a FTAI protocol may improve pregnancy success in postpartum beef cows.

Ultrasonography-accessed luteal size endpoint that most closely associates with circulating progesterone during the estrous cycle and early pregnancy in beef cows

The aim was to evaluate the associations between circulating P4 concentrations, corpus luteum (CL) size (diameter, area or volume) and blood perfusion (BP) in cows. In Experiment 1, Pearson’s correlations (P < 0.05) with P4 concentrations were observed during CL development (D8) for total area (TA; r = 0.76), luteal area (ACL; r = 0.72), total and luteal diameter (TD and DCL respectively; r = 0.46). During mid-late diestrus, there was a positive correlation (P < 0.05) only at D15 with TA and ACL (r > 0.60), TD, total volume (TV) and luteal volume (VCL; r > 0.434). During luteal regression, the correlation was only observed at D18 for ACL (r = 0.478) and D20 with several variables. In Experiment 2, CL weight and ACL had the greatest correlation with P4 (r > 0.6). In Experiment 3, TA and ACL were the variables that were most closely correlated with serum P4 concentrations at D7 in recipient cows. Correlation coefficients were greater for luteal measurements when there were compact compared with cavitary CLs. In Experiment 4, there was no correlation (P > 0.05) between P4 and any of the variables measured on D4 and D7 in recipient cows detected in estrus. On D18 to D20, all CL characteristics were correlated (P < 0.05) with plasma P4, and luteal BP and BP area were more closely (P < 0.05) correlated than ACL. In conclusion, CL perimeter area measurements had the greatest association with luteal function during CL development; whereas for BP there was a greater correlation with P4 than luteal size during luteolysis.

16 False-positive rate after anticipation of early pregnancy diagnosis for resynchronization of ovulation in Bos indicus heifers

The objective was to determine the incidence of false-positive pregnancy diagnosis following the use of colour Doppler ultrasonography 20 and 22 days after fixed-time AI (FTAI) in Bos indicus heifers submitted to resynchronization 14 days after first FTAI. In the study, 512 Nellore heifers at 24.5±0.8 months of age and body condition score of 3.4±0.1 were used beginning 14 days after FTAI. On Day 14, heifers received 50mg of short-acting progesterone (Afisterone®, CEVA, São Paulo, Brazil) and a progesterone device (Prociclar®, CEVA). Eight days later (Day 22), the progesterone device was removed. A duplex B-mode (grayscale) and pulse-wave colour Doppler ultrasound instrument (M5, Mindray, Shenzhen, China) equipped with a multifrequency linear transducer was used for the examination of luteal blood flow on Days 20 and 22. The percentage of luteal area with colour Doppler signals of blood flow at each examination was determined as previously described (Ginther 2007) and was classified as low [corpus luteum (CL) with less than 25% vascularized area], intermediate (CL with 25-75% vascularized area), or high (CL with more than 75% vascularized area). Heifers with intermediate and high luteal blood flow were diagnosed as pregnant by Doppler ultrasonography, and heifers with low luteal blood flow were diagnosed as nonpregnant. Heifers diagnosed as pregnant by Doppler ultrasonography were examined 30 days after FTAI by B-mode ultrasound examination for pregnancy diagnosis and to determine the number of false positives (heifers pregnant by Doppler ultrasonography and nonpregnant by B-mode ultrasonography). Statistical analysis was performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Pregnancy rate by Doppler ultrasonography [Day 20=60.5% (310/512), Day 22=55.3% (283/512); P=0.10] and false-positive percentage [Day 20=29.7% (92/310), Day 22=23.0% (65/2830); P=0.06] were similar on pregnancy diagnoses by Doppler ultrasonography performed 20 and 22 days after FTAI. Furthermore, in the period from 20 to 22 days after FTAI, luteolysis was verified in 5.8% (30/512) of heifers. In conclusion, anticipation of pregnancy diagnosis in 2 days by evaluation of luteal blood flow with colour Doppler ultrasonography (20 days after FTAI) does not interfere with pregnancy rate by Doppler ultrasound and percentage of false positives in Bos indicus heifers.

The Use of Power Mode Doppler Ultrasonography as a Predictive Tool of Early Pregnancy in the Mare

Human chorionic gonadotropin (hCG) has been used to induce ovulation and as a luteotrophic agent in cattle. However, the effect of hCG therapy on the functional status of the equine corpus luteum (CL) is unclear. This study aimed to characterize the hemodynamic and secretory function of early CL of mares treated with different doses of hCG at distinct stages of the estrous cycle. Mares were assigned to nine experimental groups (n = 6 mares/group) according to dose of hCG and time of treatment. A single injection of one of three different doses of hCG (750, 1,500, or 2,500 IU) was performed in one of three distinct stages of the estrous cycle: preovulatory follicle ≥35 mm, day of ovulation (D0), or 48 hours after ovulation (D2). In addition, a control group treated with NaCl 0.9% was included in the study. The end points evaluated daily from D0 to D8 were area of the CL, luteal vascularity, number of colored pixels and total pixel intensity, and concentrations of plasma progesterone (P4). No effect (P > .1) of dose or time of treatment was observed for any end point, within each day. Luteal area did not differ throughout the days (P > .1), whereas Doppler parameters and concentrations of plasma P4 presented a progressive increase (P < .05) after ovulation in all groups. Secretory function and luteal hemodynamic were not affected (P > .1) by hCG dose and time of treatment. In conclusion, hCG therapy during estrus or early diestrus, at the doses tested, did not improve P4 secretion or luteal blood flow.

Effects of extending the length of pro-oestrus in an oestradiol- and progesterone-based oestrus synchronisation program on ovarian function, uterine environment and pregnancy establishment in beef heifers.

The aim of the present study was to investigate the effects of a strategy for extending pro-oestrus (the interval between luteolysis and ovulation) in an oestrus synchronisation protocol (named J-Synch) in beef heifers on follicular growth, sexual steroid concentrations, the oestrogen receptor ERα and progesterone receptors (PR) in the uterus, insulin-like growth factor (IGF) 1 and pregnancy rates. In Experiment 1, heifers treated with the new J-Synch protocol had a longer pro-oestrus period than those treated with the conventional protocol (mean (±s.e.m.) 93.7±12.9 vs 65.0±13.7h respectively; P<0.05). The rate of dominant follicle growth from the time of progesterone device removal to ovulation was greater in heifers in the J-Synch than conventional group (P<0.05). Luteal area and serum progesterone concentrations were greater in the J-Synch Group (P<0.05) for the 12 days after ovulation. Progesterone receptor (PGR) staining on Day 6 after ovulation in the uterine stroma was lower in the J-Synch than conventional group (P<0.05), and the expression of PR gene (PGR) and IGF1 gene tended to be lower in J-Synch-treated heifers (P<0.1). In Experiment 2 (n=2349), the pregnancy rate 30-35 days after fixed-time AI (FTAI) was greater for heifers in the J-Synch than conventional group (56.1% vs 50.7% respectively). In conclusion, our strategy for extending pro-oestrus (i.e. the J-Synch protocol) significantly improves pregnancy establishment in beef heifers. This improvement was related to an increased rate of growth of the dominant ovulatory follicle, greater progesterone concentrations during the ensuing luteal phase and different uterine patterns of PGR and IGF1, which may have favoured embryo development and pregnancy establishment.

The effect of presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously, 7 d before Ovsynch, compared with Presynch-10/Ovsynch on luteal function and first-service pregnancies per artificial insemination

Pregnancy per artificial insemination (P/AI) following Ovsynch is optimized when cows ovulate to the first GnRH of Ovsynch. Fertility programs are designed to presynchronize cows to d 6 or 7 of the estrous cycle to increase the chances of ovulation of a first-wave dominant follicle to the first GnRH of Ovsynch. The hypothesis of this experiment was that simplification of a presynchronization program through the combination of PGF2α and GnRH on the same day, 7 d before Ovsynch, would allow for similar P/AI compared with Presynch-10. Lactating dairy cows (n = 432) 41 to 47 d in milk (DIM) were randomly assigned to 2 treatments within parities for first service. Control cows received Presynch-10/Ovsynch consisting of the following: PGF2α-14 d-PGF2α-10 d-GnRH-7 d-PGF2α-56 h-GnRH-16 h-AI. Treated cows received PGF2α and GnRH-7 d-GnRH-7 d-PGF2α-56 h-GnRH-16 h-AI. All cows received a supplemental injection of PGF2α 24 h after the PGF2α of Ovsynch to enhance complete luteolysis. All cows received timed AI between 75 and 81 DIM. Blood was collected to assess circulating concentrations of progesterone (P4), and the number and size of corpora lutea (CL) were recorded using ultrasonography on day of PGF2α of Ovsynch. The administration of PGF2α simultaneously with GnRH and 7 d before Ovsynch (PG+G) had similar P/AI at 28 (46 vs. 48%), 35 (43 vs. 43%), 49 (39 vs. 39%), and 77 d post-AI (38 vs. 39%) compared with Presynch-10. No differences were observed in P/AI in primiparous versus multiparous cows at 28 (52 vs. 45%), 35 (48 vs. 41%), 49 (45 vs. 37%), and 77 d post-AI (43 vs. 36%). No difference existed between treatments in percentage of cows with functional CL at PGF2α of Ovsynch, total luteal area (mm2), or serum concentrations of P4 at time of PGF2α of Ovsynch, regardless of parity. Number of CL had a tendency to be greater for multiparous PG+G vs. Presynch-10 cows (2.34 ± 0.09 vs. 2.15 ± 0.08) but not in primiparous cows (1.95 ± 0.10 vs. 1.98 ± 0.11). In summary, administering both PGF2α and GnRH on the same day, 7 d before the start of Ovsynch, appears to be a simple and effective alternative to Presynch-10 Ovsynch.

Pregnancy outcome is influenced by luteal area during diestrus before successful insemination but not by milk production level

The objective was to compare luteal area (LA), luteal blood flow (LBF), and progesterone (P4) concentration before and after artificial insemination (AI) in pregnant and open cows in the diestrus preceding insemination and during the first 21 d after insemination. A number of 119 multiparous German Holstein cows were examined using B-mode and color Doppler sonography in diestrus (15–5 d before AI) and on days 7, 14, and 21 post insemination (pi). Blood samples for determination of P4 concentration were collected at each examination. In pregnant cows, P4 was measured in the diestrus before AI and on days 7, 14, and 21 of pregnancy, and in open cows, in the diestrus preceding the first AI and 7, 14, and 21 d later. Cows were retrospectively allocated into two groups according to the mean daily milk yield in the first 42 d (high milk yield [HMY], ≥39 kg/d; low milk yield [LMY], <39 kg/d). Pregnant cows had larger LA than open cows during diestrus before insemination (5.69 ± 1.85 vs. 4.94 ± 1.66 cm2, P < 0.05), on days 7 (5.34 ± 1.83 vs. 4.52 ± 1.93 cm2, P < 0.05) and 21 pi (5.92 ± 1.60 vs. 4.97 ± 1.44 cm2, P < 0.05). On day 14 there was a tendency towards larger LA in pregnant cows (P = 0.09). Luteal blood flow (1.72 ± 0.74 vs. 1.22 ± 0.67 cm2) and P4 concentration (8.97 ± 4.37 vs. 6.49 ± 4.32 ng/mL) were higher in pregnant cows than in open cows on day 21 (P < 0.05). At day 150, 69% of the LMY cows and 56% of the HMY cows were pregnant (P > 0.05). Pregnant HMY cows had larger LA during diestrus before insemination and on days 7 and 21 pi than open HMY cows (P < 0.05) and tended to result in larger CLs on day 14 (P = 0.06). Pregnant HMY cows had higher LBF on days 14 and 21 (P < 0.05) and higher P4 concentration on day 21 than open HMY cows (P < 0.05). The main finding of this study was a larger LA during diestrus in cows that conceived in the ensuing estrous cycle compared to cows that did not conceive. Luteal area, LBF, and P4 concentration were greater in pregnant cows compared to open cows, and further studies should be conducted on the usefulness of these variables to detect open cows as early as possible after breeding. High-yielding cows appear to require larger LA, LBF, and higher P4 concentration than low-yielding cows to maintain pregnancy. In conclusion, larger LAs were established in pregnant cows in the diestrus before successful insemination regardless of their milk yield. Further studies are needed to verify whether LA in the diestrus before insemination might be used to predict probabilities in reproductive performance.

121 THREE-DIMENSIONAL ASSESSMENT OF EARLY CORPUS LUTEUM VASCULARITY IN BUFFALO (BUBALUS BUBALIS)

The aim of the study was to develop an objective method to assess the vascular flow to the early corpus luteum (CL) in buffaloes using colour Doppler ultrasound data. Our hypothesis was that 3-dimensional (3D) volumetric analysis of vascularity would demonstrate lower variability between animals compared with conventional 2-dimensional (2D) analysis of single images. Wave emergence and ovulation was synchronized in buffalo (n = 16) using prostaglandin-GnRH based protocols. Colour Doppler ultrasonography (MyLab5, 7.5-MHz linear array, colour gain 65%) was performed daily from Day −2 to 4 (Day 0 = ovulation). Video clips of the ovaries (20 s at 18–28 frames per second, AVI) were recorded by slow and uniform free-hand movement of the transducer. Day 4 CL was used for analysis of vascular area and volume. For 2D vascularity assessment, 3 images (800 × 652 pixels, RGB, BMP) of each CL (at maximum apparent vascularity) were acquired through the clip image function on the ultrasound machine and analysed by ImageJ (Fiji) software (NIH, Bethesda, MD, USA). For 3D vascularity assessment, a portion of the video clip encompassing an entire ovary was identified and exported as a series of 2D TIFF images using Videomach software. The ultrasound scale bar was used to calculate the number of pixels per millimetre and to calibrate the X (horizontal) and Y (vertical) dimensions. For 2D analyses, the CL boundary was drawn using the free-hand manual selection tool in Fiji, the area of the CL (mm2) was recorded, and the border was then enlarged by 1.5 mm to include the peripheral vascular region of the CL. The colour threshold was adjusted to select the vascular region. The 2D vascularity score was calculated as the ratio of the coloured area to the enlarged luteal area. For 3D volumetric analyses, each series of TIFF images was imported as an image sequence in Fiji and colour thresholding (similar to 2D analysis) was applied to save a second TIFF series containing luteal vascular regions (coloured areas) only. The remaining volumetric analyses were completed in Imaris software using the ovarian volume (original TIFF series) and luteal vascular volume (second TIFF series) as separate channels. The Z-dimension thickness of each image was estimated by using the dimensions of a follicle within the same ovary (Z-axis diameter = mean diameter along X- and Y-axes). Similar to 2D analyses, the volume of the CL was obtained by drawing a border along the edge of the CL, the CL border was enlarged by 1.5 mm, and a 3D vascularity score was obtained by building a surface on the luteal vascular image and calculating the vascular to luteal volume ratio. The 2D vascularity score differed from 3D vascularity score (0.21 ± 0.02 v. 0.13 ± 0.02, paired t-test P &lt; 0.01); however, variance did not differ (Bartlett’s test P = 0.32). Our initial results support the notion that the described technique of quantifying vascular volume of the corpus luteum may decrease the technical variability during image assessment and therefore better reflect the true vascularity compared with 2D image analyses. Research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

Colour Doppler Ultrasonography as a Tool to Assess Luteal Function in Santa Inês Ewes.

The aim of this study was to evaluate luteal dynamics in the Santa Inês ewes using colour Doppler (CD) ultrasonography. Oestrus was synchronized in nulliparous females (n=18), and subsequently, they were only teased (n=6) or teased and mated (n=12). Blood samples were collected daily for plasma progesterone (P4 ) concentrations. Ultrasonographic images of corpora lutea (CL) in CD mode were obtained for further analysis in its largest diameter. The CD mode allowed an early sequential monitoring of CL that was visualized by the first time 0.77±0.62days after ovulation, with luteal area 29.68±13.21mm(2) . During the luteogenesis, a progressive increase was observed, followed by a plateau of luteal area, vascularization area and plasma concentrations of P4 reaching maximum values in D11 (124.0±38.0mm(2) , 52.78 ±24.08mm(2) and 11.23±4.89ng/ml, respectively). In the luteolysis, the plasma concentrations of P4 decreased sharply, whereas luteal and vascularization area gradually. The vascularization area was positively correlated with plasma concentrations of P4 during the luteogenesis (r=0.22) and luteolysis (r=0.48). The luteal dynamics of Santa Inês ewes showed patterns similar to those observed in other sheep breeds studied. The CD ultrasonography has the potential to be used as a tool to assess luteal function in sheep.

Characterization of Luteal Blood Flow and Secretion of Progesterone in Mares Treated With Human Chorionic Gonadotropin for Ovulation Induction or During Early Diestrus

Human chorionic gonadotropin (hCG) has been used to induce ovulation and as a luteotrophic agent in cattle. However, the effect of hCG therapy on the functional status of the equine corpus luteum (CL) is unclear. This study aimed to characterize the hemodynamic and secretory function of early CL of mares treated with different doses of hCG at distinct stages of the estrous cycle. Mares were assigned to nine experimental groups (n = 6 mares/group) according to dose of hCG and time of treatment. A single injection of one of three different doses of hCG (750, 1,500, or 2,500 IU) was performed in one of three distinct stages of the estrous cycle: preovulatory follicle ≥35 mm, day of ovulation (D0), or 48 hours after ovulation (D2). In addition, a control group treated with NaCl 0.9% was included in the study. The end points evaluated daily from D0 to D8 were area of the CL, luteal vascularity, number of colored pixels and total pixel intensity, and concentrations of plasma progesterone (P4). No effect (P > .1) of dose or time of treatment was observed for any end point, within each day. Luteal area did not differ throughout the days (P > .1), whereas Doppler parameters and concentrations of plasma P4 presented a progressive increase (P < .05) after ovulation in all groups. Secretory function and luteal hemodynamic were not affected (P > .1) by hCG dose and time of treatment. In conclusion, hCG therapy during estrus or early diestrus, at the doses tested, did not improve P4 secretion or luteal blood flow.

176 BLOOD FLOW TO THE CORPUS LUTEUM AND PREOVULATORY FOLLICLE AFTER OVULATION INDUCTION DURING FIRST VERSUS SECOND WAVE IN WATER BUFFALO

The objective of the study was to compare the blood flow to the corpus luteum (CL) and the preovulatory follicle in dairy buffalo (Bubalus bubalis) when ovulation was induced during the first (low to increasing progesterone levels) versus the second (luteal progesterone levels) follicular wave. We hypothesised that the wall of the first-wave dominant follicle will be less vascular compared with that of the second-wave follicle. The study was conducted during the summer months in Punjab, India. Ovulation was synchronized with prostaglandin F2α (PGF) IM followed by gonadotropin-releasing hormone (GnRH) IM 48 h later (Day 0) and buffaloes were randomised to first wave (FW; n = 6) and second wave (SW; n = 7) groups. FW group was given PGF on Days 6.5 and 7, and GnRH on Day 9.5 followed by AI (14–16 h after GnRH). The SW group was given GnRH on Day 7 (to induce ovulation of first-wave dominant follicle without luteolysis and synchronous emergence of next wave), PGF on Days 13.5 and 14, GnRH on Day 16.5 followed by artificial insemination. Transrectal colour Doppler ultrasonography (MyLab5 Vetwith 7.5 MHz transducer, Esaote S.p.A, Genoa, Italy) was performed daily and 20-s cineloops of each ovary were recorded under standardized gain controls. Images from the cineloops were processed using Fiji (ImageJ, National Institutes of Health, Bethesda, MD, USA) to calculate the area of blood flow (coloured area = vascular area, grey scale area = tissue area, and their ratio) for the preovulatory follicle (on the day before ovulation) and luteal tissue (on the day of PGF injection and 4 days post-ovulation). Data were analysed by t-test from the animals that ovulated one day before (n = 3) or the day of AI (n = 6) and had a functional CL at day 5 post-AI (FW n = 5, SW n = 4). FW follicles ovulated on 8.6 ± 0.3 days from wave emergence compared with SW follicles on 10.0 ± 0.6 days (P &lt; 0.05) but were similar in size (i.e. follicular area on the day before ovulation did not differ between groups; P = 0.5). There was no difference in the blood flow area in the wall of preovulatory follicles (P = 0.4). Vascular area of follicles was strongly correlated with their diameter (r = 0.87). Follicles &gt;13.5 mm in diameter had more blood flow in their wall than smaller follicles (P &lt; 0.01). FW had a tendency (P = 0.07) for smaller luteal area on the day of PGF treatment (FW = 171 ± 24 mm2; SW = 332 ± 81 mm2) and tended (P = 0.06) to have less vascular area in the CL compared to SW group (FW = 30 ± 6 mm2; SW = 67 ± 17 mm2). There was no difference (P = 0.5) between the groups for vascular to CL area ratio. The area of luteal tissue and blood flow to the CL at Day 4 post-ovulation did not differ between the groups (P = 0.4). The diameter of the preovulatory follicle (11.6–15.7 mm) was not correlated with the cross-sectional area of developing CL at Day 4 post-ovulation (r = 0.09). In conclusion, vascularity to preovulatory follicles originating from the first wave v. second wave did not differ and preovulatory follicles ≥13.5 mm were more vascular than smaller follicles. Research was funded by NSERC; the first author was funded by scholarships from WCVM and GADVASU.

11 STRATEGIES TO MODULATE THE PERI-OVULATORY ENDOCRINE MILIEU BY CONTROLLING PROGESTERONE CONCENTRATIONS PRE- AND POST-INSEMINATION IN BEEF COWS

Recent evidence indicates that progesterone (P4) secretion from corpus luteum (CL) is affected by preovulatory follicle (POF) size and that increased circulating P4 during early diestrus has a positive effect on embryo development and pregnancy rates in cattle. Despite the embryotrophic effects, early administration of P4 may impair CL growth and anticipates luteolysis. Owing to develop favourable peri-ovulatory endocrine milieus to pregnancy success, we evaluated in beef cows (1) the effect of CL presence during dominant follicle growth on follicular vascularization, size and ovulation rate and (2) the effects of POF size and supplementation of long-acting P4 post-AI on CL development and regression. Sixty-six Nelore cows received an intravaginal P4 device and an injection of oestradiol benzoate on Day 10 (5–10 days post-oestrus), and were split to receive sodium cloprostenol [prostaglandin F2α (PGF2); large follicle (LF); n = 31) or not [small follicle (SF); n = 35]. Devices were removed and PGF2 was injected on Day 1.75. Ovulation was induced with a gonadotropin-releasing hormone (GnRH; buserelin acetate) on Day 0. Cows were inseminated twice on Days 0.5 and 1. Cows that ovulated were assigned to receive placebo (LF/control group, n = 14; and SF/control group, n = 9) or 150 mg of long-acting P4 on D4.5 (LF/P4 group, n = 13; and SF/P4 group, n = 12). Colour-Doppler ultrasound scanning was done on Days 10, 6, 2, 0.5, 1, 1.5, 4.5, 7.5, and 15.5. Structural luteolysis was detected by 25% decrease in CL area and 50% in luteal blood flow of subsequent scanning. Data were analysed for the main effects of POF size and supplementation and their interaction using the PROC MIXED procedure of SAS (SAS Institue Inc., Cary, NC, USA). Follicle diameter (mm) on Day 2 (11.4 ± 0.4 v. 8.4 ± 0.3) and POF (14.1 ± 0.3 v. 12.5 ± 0.3) were greater (P &lt; 0.05) in LF group. Percentage of luteal blood flow in follicle wall was greater (P &lt; 0.05) on Day 0.5 (33.9 ± 3.4 v. 22.8 ± 3.6) and 1 (48.9 ± 4.8 v. 28.3 ± 4.3) in the LF group. Ovulation rate was lower (P &lt; 0.05) in the SF group (60%, 21/35) than in the LF group (90%, 28/31), suggesting that a functional CL during dominant follicle growth is less favourable to pregnancy success due to 30% reduction in fertilizing potential of non-ovulated follicles. For luteal area (cm2), an effect (P &lt; 0.05) of POF size was detected on Day 4.5 (1.7 ± 0.1 v. 1.3 ± 0.1), 7.5 (2.8 ± 0.2 v. 2.2 ± 0.1), and 15.5 (2.5 ± 0.1 v. 2.2 ± 0.2) in the LF and SF groups, respectively. Luteal blood flow was greater (P &lt; 0.05) in the LF groups (40.4 ± 1.7 and 47.6 ± 3.4) than in the SF groups (35.7 ± 1.5 and 37.9 ± 4.1) on Day 4.5 and 15.5, respectively. This indicates that larger and more vascularized POF may be associated with greater CL vascularization in addition to greater CL size. The frequency of cows with early luteolysis did not differ (P &gt; 0.1) between P4-treated (5/25) and placebo-treated (1/23) cows. We suggest that the P4 supplementation did not severely induce early luteolysis in inseminated cows and that the P4-embryotrophic effects may down-regulate the mechanisms involved in the earlier luteolytic process caused by greater exposure of uterus to P4. Support for this study was provided by the São Paulo Research Foundation (FAPESP, Sao Paulo, Brazil) and Ourofino (Cravinhos, SP, Brazil).

The effect of hCG, administered in diestrus, on luteal, ovarian and uterine blood flow, peripheral progesterone levels and pregnancy rates in mares

Although advances in the management of brood mares have led to an increase in conception rates, early embryonic loss is still a major concern in equine practice. However, administration of hCG in diestrousmares has been reported to increase pregnancy rates and progesterone production. The aim of this study was to analyze the effects of a postovulatory hCG-treatment on luteal, ovarian and uterine blood flow, peripheral serum progesterone concentrations and pregnancy rates in the mare. Thirty-four mares were inseminated, 17 with cooled and 17 mares with frozen semen, and randomly assigned to three experimental groups after ovulation (D0). Group 1 (n 1⁄4 12) served as untreated control, Group 2 (n1⁄411) received a single intravenous injection of 2500 IU hCG on D5, and Group 3 (n 1⁄4 11) got an analogous hCG-application on D 11 of the cycle. On D0 during the first 12 h post ovulation colour Doppler ultrasonography was performed every 4 h, and afterwards in 12-h-intervals until D3 in all groups. The control group was examined once daily until D13. Mares in the treatment groups were examined once daily until the hCG-administration. Thereafter ultrasonographic examinations were performed every four hours for the first 12 h and in 12-h-intervals for the following three days. The total as well as the perfused luteal area, and the blood flow

Progesterone concentration when the future ovulatory follicle and corpus luteum are located in ipsilateral or contralateral ovaries in heifers

Three studies were done on the effects of ipsilateral location (same ovary) versus contralateral location (opposite ovaries) of the future ovulatory follicle and CL in heifers. The numbers of heifers for the ipsilateral and contralateral groups, respectively, were: experiment (Exp) 1 (N = 4 and 4), Exp 2 (N = 6 and 4), and Exp 3 (N = 5 and 10). In the Exps with available data (Exp 2 and 3), the interval between ovulation and the end of luteolysis was significantly shorter in the ipsilateral group than in the contralateral group (Exp 2: 16.8 ± 0.3 vs. 19.8 ± 1.7 days; Exp 3: 16.9 ± 0.2 vs. 19.7 ± 0.9 days). In Exp 3, the interovulatory interval was shorter (P < 0.01) in the ipsilateral group (20.1 ± 0.4 days) than in the contralateral group (22.7 ± 0.7 days), but the interval from the end of luteolysis to ovulation was not altered significantly. Circulating progesterone (P4) concentration for 33 hours normalized to the beginning of luteolysis (Exp 1) and on Days 16 to 20 (Day 0 = ovulation; Exp 3) was significantly lower in the ipsilateral group than in the contralateral group (Exp 1: 3.7 ± 0.2 vs. 4.8 ± 0.3 ng/mL; Exp 3: 1.7 ± 0.4 vs. 5.9 ± 0.4 ng/mL). Area (cm2) of the CL and percentage of CL with color Doppler signals of blood flow were lower and resistance index for a CL arteriole was greater in the ipsilateral group (Exp 3). The decreased P4 concentration in the ipsilateral group began by Day 16, but the decreased luteal area and vascular perfusion were not detected until Days 17 or 18. Results supported the hypothesis that the ipsilateral location of the future ovulatory follicle and CL was associated with lower P4 production and a shorter interovulatory interval.

Correlations between ultrasonographic characteristics of corpora lutea and systemic concentrations of progesterone during the discrete stages of corpora lutea lifespan and secretory activity in cyclic ewes

Associations between physical characteristics and functionality of corpora lutea (CL) have previously been reported in monovulatory species, albeit several studies in cattle and humans have refuted the existence of temporal relationships between CL size, echotexture and serum progesterone (P(4)) concentrations. The main objective of the present study was to examine whether or not there were correlations between ultrasonographic image attributes of CL and systemic concentrations of P(4) during the discrete stages of the luteal phase in two breeds of sheep differing in ovulation rates (non-prolific Western White Face [WWF] ewes and prolific Finn [F] sheep). Transrectal ovarian ultrasonography utilized a 7.5-MHz linear-array transducer connected to a portable scanner (Aloka SSD-500) and the images were analyzed using commercially available image analytical software (Image ProPlus(®)) validated for the present application in sheep. The correlations were assessed using the Pearson's Product Moment (PPM) analysis and also, to increase the accuracy of statistical tests, the analysis of covariance (ANCOVA), with the number of CL as a co-factor. In WWF ewes, serum concentrations of P(4) correlated significantly with the total luteal area (TLA) during the CL growth phase (days 3-6; day 0 = ovulation) and functional luteolysis (days 12-15), and with numerical pixel values (NPVs--pixel intensity) during luteolysis; the results obtained by using two different statistical methods were generally similar. In prolific F ewes, serum P(4) concentrations were directly correlated with TLA during CL growth (days 3-6; ANCOVA), functional luteolysis (days 13-14; PPM), and structural CL regression (days 11-14; PPM and ANCOVA), and with NPVs during functional luteolysis (PPM and ANCOVA). We concluded that systemic P(4) concentrations could only be accurately predicted from the changes in luteal area during CL growth and regression, and from NPVs during luteolysis, in both prolific and non-prolific ewes, but the changes in size and echotexture of the luteal glands at mid-cycle were not indicative of serum P(4) concentrations in sheep.

Patterns of Corpora Lutea Growth and Progesterone Secretion in Sows with Thrifty Genotype and Leptin Resistance due to Leptin Receptor Gene Polymorphisms (Iberian Pig)

The current study aimed to compare luteal function, as measured by corpora lutea dynamics and progesterone secretion, in 10 sows with obesity/leptin resistance genotype (Iberian pig) and 10 females of lean commercial crosses (Large White × Landrace). In all the animals, the oestrous cycle was synchronized with progestagens, and ovulation was induced by exogenous gonadotrophins. Thereafter, number and size of follicles and plasma oestradiol concentration were determined at oestrus detection, and number and size of corpora lutea and progesterone concentration were evaluated from Day 4 to 12 of the cycle. There were no differences between genotypes in follicle population and oestradiol concentration, and ovulation rate (15.2±1.3 in Iberian vs 12.7±1.8 in LWxL sows); however, there was a higher percentage of Iberian than control sows showing luteal cysts (66.7% vs 30%, respectively; p<0.05). In both breeds, both total luteal area and plasma progesterone concentration grew linearly from Day 4 to 8 (p<0.01) and remained more stable between Days 8 and 12, without significant differences between genotypes. In conclusion, current study supports that ovulatory processes and luteal functionality are not the main limiting factors for prolificacy in a pig model of leptin resistance and obesity.

Increase in progesterone and luteal blood flow without a luteolytic response after prostaglandin F 2α treatment in early luteal-phase heifers

Acute changes in circulating progesterone concentration and luteal blood flow in heifers after a conventional dose of prostaglandin F 2α (PGF; 25 mg dinoprost, i.m.) were compared between the early luteal phase (Day 3) and midluteal phase (Day 10; Day 0 = ovulation), using four groups (Day-3 control, Day-3 PGF, Day-10 control, and Day-10 PGF; n = 6 heifers/group). Blood samples were collected at 0, 2, 5, 10, 15, 30, 60, and 120 min (0 min = treatment). Percentage of luteal area with color-Doppler blood-flow signals was estimated at 0, 10, and 30 min. In the Day-3 and Day-10 PGF groups, progesterone increased to a peak at 15 min. In the Day-3 PGF group, progesterone decreased to the pretreatment concentration by 60 min but did not decrease to below the pretreatment concentration during the 2-h experimental period. In the Day-10 PGF group, progesterone decreased to below pretreatment concentration by 30 min, indicating a luteolytic response. In the Day-3 and Day-10 PGF groups, luteal blood flow increased within 10 min and remained elevated until the last examination at 30 min. The absence of a decrease in progesterone to below pretreatment concentrations in the Day-3 PGF group indicated that luteolysis does not necessarily follow a transient increase in progesterone and a concomitant increase in luteal blood flow. The immediate transient increase in progesterone and an increase in luteal blood flow without a subsequent decrease in progesterone to below pretreatment concentrations after PGF treatment in early luteal-phase heifers are novel findings.

Temporal gene expression in equine corpora lutea based on serial biopsies in vivo1

A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares with normal estrous cycles and 1 mare with a retained CL. The biopsy procedure did not adversely affect luteal size or function, as measured by luteal area and serum concentrations of progesterone. Real-time reverse-transcription PCR was used to quantify steady state mRNA concentrations in each tissue sample obtained. Mean abundance of steroidogenic acute regulatory protein (StAR) mRNA was not different (P = 0.102 to 0.964) on any of the sampling dates, but a trend for mRNA encoding StAR to decrease between d 12 and 14 (P = 0.10) was observed. Values for mRNA encoding StAR were positively correlated to serum concentrations of progesterone on d 5 (R = 0.95; P = 0.05) and 14 (R > 0.99; P < 0.01). Steady-state abundance of mRNA for 3β-hydroxysteroid dehydrogenase, Δ 5-Δ 4 isomerase (3β-HSD) declined between d 12 and 14 (P = 0.15). There were positive correlations between mRNA for 3β-HSD and concentrations of progesterone on d 5 (R = 0.94; P = 0.06) and 12 (R > 0.99; P = 0.05). No difference was detected in abundance of mRNA encoding cyclooxygenase-2 (cox-2; P = 0.340 to 0.840) or caspase-3 (P = 0.517 to 0.882) between any of the sampling dates. A successful luteal biopsy procedure was developed that did not negatively affect luteal function, and abundance of mRNA encoding StAR, 3β-HSD, cox-2, and caspase-3 was characterized in luteal biopsy tissue collected on d 2, 5, 12, and 14 of the estrous cycle in the mare.

Luteal dynamics in goats: morphological and endocrine features

The aim of this study was to establish the morphologic and endocrine characteristics of luteal dynamics in goats. It was used Toggenburg female goats that showed natural estrus in a 48-hour interval. After estrus, ultrasonographic evaluations of the ovaries were daily performed during 21 days using a portable device (5MHz probe). Blood sample was collected for plasma progresterone (P4) determination. Corpora lutea were detected for the first time on day 5 and progressively increased in size until D9 (1.26 ± 0.08 cm²), with no variation on subsequent days. In females with one ovulation, the first visualization of the corpora lutea was earlier than in those with multiple ovulation (4.54 ± 0.18 vs 5.74 ± 0.25 days). At the moment of the first visualization, luteal area was smaller in animals with single ovulation. Plasma P4 concentration progressively increased until day 9 and it did not show significant increase until luteolysis, characterized by a sharp decrease in P4 concentration, reaching values below 1 ng/mL in 24 hours. The luteal area slowly and gradually decreased in size. It was observed a significant positive correlation between P4 concentration and area during luteogenesis and luteolysis (r = 0.63 and r = 0.50, respectively). When corpus luteum reached its maximum size (D9), female with more than one corpora lutea, with a greater luteal tissue area, did not show P4 concentration higher than those with one ovulation (5.92 ± 0.59 vs 7.04 ± 0.79 ng/mL). These results show that luteal dynamics in Toggenbur goats follow a similar pattern to those observed in other goat breeds and luteal tissue growth was positively correlated with corpora lutea functionality.

Assessment of luteal function in goats by ultrasonographic image attribute analysis

The aim of this study was to evaluate the potential of luteal echotexture (mean pixel value and heterogeneity), as a tool for assessing luteal function during different phases of the estrous cycle in Toggenburg goats. Sonographic evaluations of the ovaries were performed daily in nulliparous goats ( n = 21), using a 5 MHz linear rectal probe, commencing at estrus (day 0). Blood samples were collected daily for plasma progesterone RIA and images recorded on VHS tape and then digitized in TIFF format at a resolution of 1500 × 1125 pixels. A representative elementary area (REA) of 5625 pixels (0.31 cm 2) of these images was analyzed using custom-developed software, for mean pixel value and heterogeneity. Mean plasma progesterone, luteal area and pixels all reached maximum values at approximately days 13 and 14, during luteogenesis. Luteolysis was characterized by an abrupt decrease in blood progesterone concentration following ovulation, and a gradual decline in luteal area and pixel values. The luteal tissue area was positively correlated with plasma progesterone concentration during both luteogenesis ( r = 0.63; P < 0.05) and luteolysis ( r = 0.50; P < 0.05). Weak correlations were recorded between the mean pixel value and luteal tissue area during luteogenesis ( r = 0.34; P < 0.05) and luteolysis ( r = 0.26; P < 0.05). Similarly, weak correlations between the mean pixel value and plasma progesterone concentration were recorded during luteogenesis ( r = 0.24; P < 0.05) and luteolysis ( r = 0.37; P < 0.05). The pixel heterogeneity was not correlated with luteal tissue area or the plasma progesterone concentration at any stage of the estrous cycle. The results show the association between the corpus luteum echotexture and steriodogenic function to be weak and the present ultrasound technology, to have limited potential in evaluating luteal function in goats.