Corpus Luteum Size Research Articles

Ovarian Follicular Dynamics, and Hormonal and Biochemical Profile in Post-Pubertal and Postpartum Jaffrabadi Buffaloes

Jaffrabadi buffalo is one of the heaviest buffalo breeds of the world (800 kg) and is known for poor reproductive efficiency. There is a scarcity of normal physiological data regarding reproductive parameters of Jaffrabadi buffalo. The primary objective of this study was to elucidate ovarian follicular dynamics, hormonal and biochemical profiles in 10 post-pubertal heifers (n=5 each of age 42±2 and 48±2 months) and five >90 days postpartum lactating buffaloes. Ultrasound monitoring of ovaries of animals was done at weekly interval and all follicles with diameter less than 4 mm (small follicles), 4-8 mm (medium follicles) and greater than 8 mm (large follicles) as well as CLs identified were recorded. Blood samples were also collected soon after each USG from all animals for assay of hormonal and biochemical profiles. The findings revealed that the average number of small, medium and large follicles, total number of follicles per ovary as well as diameters of largest and subordinate follicles varied greatly among periods between three groups. The size of corpus luteum ranged from 13.7 to 21.2 mm, 13.7 to 23.9 mm and 15.3 to 22.7 mm, respectively, in Group-I, II and III. In the first week of study, significantly higher (p less than 0.05) average numbers of small follicles in Group-I and III, and medium follicles and total numbers of follicles in Group-III were observed as compared to other groups. The diameter of largest follicles and subordinate follicles were non-significantly different among all groups at all periods of the study. Plasma estrogen levels were significantly higher (p less than 0.05) in Group-I as compared to Group-III and Group-II on first and second week, however, it increased significantly (p less than 0.05) in Group-II and I on fourth week of study period. Plasma insulin concentrations were significantly (p less than 0.01) lower in buffaloes (Group-III) than in heifers (Group-I and II) throughout the study period, while plasma total cholesterol and total protein levels were higher in Group-III on 3rd to 5th weeks of study. Further, there were no significant differences in the plasma FSH, LH, progesterone and blood glucose levels in animals of three groups between periods at any of the week. The ultrasonographic and endocrine profile suggested establishment of cyclicity with silent ovulation. However, the behavioural estrus was still not manifested by these post-pubertal heifers and postpartum lactating buffaloes. These results are helpful to diagnose anovulatory conditions and/or true anestrus and initiate estrus synchronization protocols for early conception in Jaffrabadi buffaloes.

Easiest and Accurate Confirmation of Early Pregnancy during Autopsy

One of the causes for suicide in young and unmarried females is relationship without wed-log which turns into pregnancy. Due to failure of relationship or to hide the condition and guilt, they commit suicide. Sometimes during autopsy, misdiagnosis of pregnancy in absence of pregnancy causes embarrassing and controversial scenarios to families. However, on the other side, often pregnancy findings are not detected in early pregnancy which leads escape of accused from punishment. Therefore, an accurate test of pregnancy is essential. In prevailing practice, size of uterus and corpusluteum are increased during pregnancy, which leads to definitive diagnosis. Nonetheless in early pregnancy, size of uterus and corpusluteum doesn’t increase considerably and findings are usually untraced. The proposed method is easy and confirmatory test for the same. It is done by examining either urine or blood serum for hCG (Human Chorionic Gonadotropin) of deceased by a hCG strip. Test result shows positive if deceased aborted pregnancy within 15 days of death.

39 Concentration of obestatin in different stages of ovarian activity in cattle

The objective of the present study was to measure serum levels of obestatin by ELISA with the purpose of establishing parameters and being able to use it as an endocrine marker for the selection of reproductive characteristics of milk and beef cattle. In test 1, 15 Creole cows with genetic tendencies of Charolais, Angus, Brangus, and Gelbvieh were sampled in diestrus and 15 in proestrus, determining their structures with an Aloka Prosound ultrasound with a 7.5-MHz linear transducer. In test 2, 16 Holstein cows were sampled in 2 different ovarian stages, diestrus and proestrus. The standardized technique was a bovine obestatin 96-well ELISA kit from My Biosource Inc. The data were analysed by measuring central tendency and linear regression, where the average levels of obestatin were related to the corpus luteum size and size and number of follicles in milk and beef cattle. We found average levels of 556.99 and 453.35 pg for beef and dairy cattle, respectively (P<0.05). We also found a significant difference (P<0.05) in obestatin concentrations depending on the size of the follicles (751.74, 615.33, and 357.39 pg for follicles of 15, 10, and 5mm, respectively). We found a significant difference in concentration of obestatin (644.53, 550.69, and 455.88 pg; P<0.05) for corpus luteum sizes of >20mm, 10–20mm, and <10mm, respectively. In dairy cattle, obestatin levels were also evaluated at different stages of the cycle, and we found concentrations of 414.79 pg in diestrus and 530.48 pg in proestrus (P<0.05). We conclude that obestatin concentration is positively associated with a greater number and size of follicles as well as with larger corpus luteum because its effect on cell proliferation interacting positively with high levels of progesterone in the blood. We found higher concentrations in beef cattle than in dairy cattle.

201 Effects of feeding vitamin and mineral and (or) energy supplements to beef heifers during the first 83 days of gestation on progesterone concentrations, corpus luteum size, and fetal body measurements

Abstract Our objectives were to determine the effects of feeding vitamin and mineral (VTM) and (or) energy (NRG) supplements to beef heifers during the first 83 days of gestation on concentrations of progesterone (P4), corpus luteum (CL) size, and fetal body measurements. Crossbred beef heifers (n = 35; initial BW = 359.5 ± 7.1 kg) were blocked by weight and assigned to one of four treatments (n = 8 or 9/group) in a 2 × 2 factorial arrangement with main effects of vitamin and mineral (NoVTM or VTM) and energy (NoNRG or NRG) supplements. A basal total mixed ration was fed once daily with VTM and NRG top-dressed. The VTM treatment (113 g•heifer-1•d-1) was initiated 71 to 148 d before AI. At AI, heifers were bred to a single sire, and NRG was initiated with target gains of 0.28 kg/d for NoNRG and 0.79 kg/d for NRG. Serum was collected on d 14, 28, 42, 56, 70, and 83 after AI and analyzed for. Gravid reproductive tracts were collected by ovariohysterectomy on d 83 ± 0.27 of gestation, and fetuses were dissected. An NRG × day interaction (P = 0.006) was observed for P4, with concentrations similar on d 14 to 56, a tendency (P = 0.09) for divergence at d 70, and greater (P = 0.002) concentrations for NRG on d 83 compared with NoNRG heifers. Furthermore, NRG heifers had greater CL weights than NoNRG (P = 0.003). Fetuses from NRG dams had greater (P = 0.009) femur weights than fetuses from NoNRG. Fetal liver weight was greater (P = 0.05) from dams fed VTM than NoVTM. Overall, providing NRG supplements during early gestation resulted in heavier CLs, that produced more P4, and greater fetal femur growth, whereas providing VTM supplements enhanced fetal liver growth.

153 Do pre and postnatal nutrition interact to program reproductive phenotype in sexually mature heifers?

Abstract Objectives were to test the hypothesis that nutritional extremes during pregnancy in the bovine female negatively affect phenotypic characteristics of the estrous cycle of sexually mature offspring, and to determine whether these effects interact with postnatal diets during the juvenile period. Beginning at 90 d of pregnancy, Brangus and Braford (n = 108) beef cows with a female fetus were fed to achieve body condition scores of 7.5–8 (H, obese), 5–5.5 (M, moderate) or 3–3.5 (L, very thin) by the start of the third trimester. Heifer offspring were weaned and fed to gain at either a high (H; 1 kg/d) or low (L; 0.5 kg/d) rate between 4 and 8 months of age, then fed a common growth diet until puberty. Estrous cycles of a subgroup (n = 53) were synchronized using 2 injections of prostaglandin F2α(PGF). Transrectal ultrasonography was employed during the follicular phase of two estrous cycles to evaluate antral follicle count(AFC), rate of growth and size of the ovulatory follicle, size of corpus luteum(CL), and endometrial thickness. Blood samples were collected daily for assay of plasma concentrations of progesterone(P4) and estradiol-17β(E2) by RIA. Preliminary data (n = 35) were analyzed using ANOVA and mixed model procedures. Least Squares Means estrous cycle length (20.9 ± 0.2 days), AFC (19.7 ± 1.0), follicular growth rate (0.81 ± 0.11), ovulatory follicle size (12.8 ± 0.2 mm), CL size (16.8 ± 0.6 mm), and endometrial thickness (14.6 ± 0.3 mm) did not differ among treatments. Heifers in postnatal H group tended (P < 0.1) to have a greater area (arbitrary units) under the E2 curve (12.6 ± 0.6) than postnatal L (11.3 ± 0.5) during the follicular phase. Normalized AUC for P4 was greater (P < 0.03) in L/H (56.94 ± 3.22) than both M/H (42.98 ± 3.61) and L/L (43.66 ± 3.22) groups. Despite marked pre- and postnatal nutritional contrasts, independent and interactive effects of nutritional treatments have been of minor significance or undetectable to date.

Diestrous Ovulations in Pregnant Mares as a Response to Low Early Postovulatory Progestogen Concentration.

Simple SummaryDuring early pregnancy in mares, progestogen is synthesized by the primary corpus luteum, which is the only source of progestogen until endometrial cup and accessory corpus luteum formation, from day 36 of pregnancy onwards. In the present study, we investigated the hormonal profile (gonadotrophin and progestogen concentrations) of 11 mares after experimental reduction of primary corpus luteum function. Two pregnancies of each mare were assigned to the control and treatment groups, respectively, and were analyzed until day 34. Low plasma progestogen concentration caused by the treatment reduced the negative feedback on the hypothalamic-pituitary axis, stimulating gonadotropin release, and luteal tissue response. Progestogen concentration restoration soon after treatment suggests a rebound effect and the resurgence of luteal function. In addition, diestrous ovulation was observed between days 11 and 15 in five treatment pregnancies (5/11), but none of the controls (0/11). Although the total luteal area increased after diestrous ovulations, corpus luteum size was not correlated to progestogen secretion. Results suggest that diestrous ovulations during early pregnancy in mares may reflect low progestogen concentrations in the early postovulatory period. Spontaneous prolongation of the luteal phase has been described in horses, but the underlying causes are still unclear. The present study investigated details of gonadotrophin and progestogen secretion in pregnant mares (n = 11) with or without experimentally reduced early postovulatory luteal function. From days 0 to 3 after ovulation, they were treated with the prostaglandin F2α (PGF2α) analogue cloprostenol or left untreated. After conceptus collection on day 34, they were assigned to the opposite treatment. Mares were affiliated to the group primary corpus luteum (n = 6) or diestrous corpus luteum (n = 5) depending on diestrous corpus luteum (CL) detection in the PGF pregnancy. For statistical comparisons, a p-value < 0.05 was significant. There was an effect of treatment (p < 0.01), but not of group on progestogen concentration. The concentration of LH was higher in PGF-treated than in untreated pregnancies (p < 0.05), but did not differ between groups. The FSH concentration did not differ between groups nor treatments. The total luteal tissue area was greater in mares with a diestrous ovulation during the PGF treatment pregnancy. Low progestogen concentration in the early postovulatory phase diminish the negative feedback on the hypothalamic-pituitary axis in early pregnancy and, thus, stimulate a luteal tissue response. Detection of secondary CL at the time of pregnancy examination in mares may reflect that early post-ovulatory progestogen concentrations were low.

100 Young Scholar Presentation: Energy and Mineral Supplementation Strategies for Beef Cattle Grazing the Northern Great Plains

Abstract Three experiments were conducted to evaluate supplementation strategies for beef cattle grazing in the Northern Great Plains. In experiment 1, 28 cow-calf pairs had access to electronic feeders (SmartFeed system; C-Lock Inc., Rapid City, SD) that monitored individual vitamin and mineral supplement (VTM) intake. Cows had greater VTM intakes (81.1 ± 8.2 g/d; P &amp;lt; 0.01) than calves (44.2 ± 8.6 g/d) but both attended feeders a similar (P = 0.71) proportion of days. Furthermore, we noted greater (P &amp;lt; 0.01) concentrations of Se, Cu, and Co in livers of HIGH intake cows compared to LOW intake cows. Experiment 2, we used the SmartFeed system to regulate intake of individual heifers (n = 60) in a group pasture scenario assigned to control (CON), VTM, or energy supplement (NRG) treatments, and evaluated supplement intake, and concentrations of liver mineral and metabolites. Though no differences were observed in BW and ADG, treatments that provided VTM enhanced liver concentrations of Se, Fe, Cu, and Co (P &amp;lt; 0.05). No differences (P = 0.85) were observed in concentrations of NEFA; however, NRG heifers had greater (P = 0.01) glucose concentrations than CON and VTM heifers. Experiment 3 objectives were to determine the influence of feeding VTM and NRG supplements to beef heifers (n = 35) during the first 84 days of pregnancy on concentrations of progesterone (P4), corpus luteum (CL) size and fetal body measurements. Providing NRG during early gestation resulted in heavier CLs (P = 0.003) that produced more P4 (P = 0.002) and greater fetal femur growth (P = 0.009) whereas providing VTM enhanced fetal liver growth (P = 0.05). Overall, we were able to successfully monitor and control individual mineral intake, corroborated those differences in liver mineral concentrations, and observed alterations in fetal growth after mineral and energy supplementation.

Effect of GnRH 7 Days Before Presynchronization With Simultaneous PGF2α and GnRH on Reproductive Outcomes in Holstein Dairy Cows.

We evaluated if an additional GnRH injection 7 days before pre-synchronization with simultaneous PGF2α and GnRH (PG+G) would improve responses to presynchronization, synchronization, and pregnancy per AI (P/AI). We hypothesized that administering GnRH 7 days before PG+G would increase ovulation and corpus luteum (CL) presence at the PG+G, improve response to OvSynch treatments and P/AI. Holstein cows were blocked by parity and randomly assigned to either a PG+G (Control, n = 205); or to GnRH followed 7 days later by PG+G (ExtG, n = 201). At enrollment, Control was left untreated, whereas ExtG received GnRH. Seven days after enrollment, Control and ExtG received PG+G followed by OvSynch 7 days later (GnRH, 7 days PGF2α, 56 h GnRH, 16 h timed AI). Ovarian dynamics were assessed using ultrasonography in a subset of cows (n = 53 for Control; and n = 50 for ExtG) at each treatment, except the 2nd GnRH of OvSynch. Pregnancy diagnosed at 32- and 67-days post AI. Ovulation at enrollment tended (P = 0.06) to be higher for ExtG, but ovulation was not different at PG+G (P = 0.41) and first GnRH of the OvSynch (P = 0.25). There was a tendency (P = 0.08) for ExtG to have larger CL than Control at PGF2α of the OvSynch. There were no differences in CL and follicle sizes in any other treatment point assessed. There were no differences (P = 0.12) in luteolysis between treatments after PG+G. Overall P/AI was similar between treatments on Day 32 (Control = 33.0% vs. ExtG = 34.6%, P = 0.75) and 67 (Control = 31.8% vs. ExtG = 32.5%, P = 0.29) post AI. There was a tendency for an interaction between treatment and parity (P = 0.09) for P/AI at day 67 post-AI. In multiparous cows, ExtG tended to have greater P/AI than Control, whereas, in primiparous cows Control tended to have greater P/AI than ExtG at day 67 post-AI. In conclusion, the effects of GnRH 7 days before PG+G presynchronization lead to positive and negative tendencies, respectively, in multiparous and primiparous cows for P/AI at day 67 post-AI and needs further investigation.

Hydroalcoholic extract and seed of Foeniculum vulgare improve folliculogenesis and total antioxidant capacity level in F1 female mice offspring

BackgroundFoeniculum vulgare (fennel) is traditionally suggested for the fertility improvement in Iranian lore due to its antioxidant and phytoestrogen compounds. The present study aimed to investigate the effects of fennel seed and its hydroalcoholic extract on the serum total antioxidant capacity (TAC) and folliculogenesis in offspring exposed to either of the treatments in utero and 56 days after birth (PND 56).MethodsPregnant NMRI mice were randomly divided into 5 groups of 7: extract-treated groups received 500 and 1000 mg/kg/day fennel extract (FE), seed-treated groups received 500 and 1000 mg/kg/day fennel seed (FS), and the control group (CTL) received no treatment. The treatments started from pregnancy day 1 and continued until PND 56. Body and right ovary weights and ovary dimensions were recorded. Hematoxylin and eosin stained ovary sections were prepared to calculate the proportion of different follicles. The level of TAC in the serums was also measured by fluorescence recovery after photo bleaching.ResultsA marked rise in the body and ovary weights of treated mice was observed compared to the CTL group. The mean number of primordial, primary, pre-antral, and pre-ovulatory follicles as well as corpus luteum size in the treated offspring was significantly higher compared to those of CTL offspring. The atretic follicle number was nonsignificantly lower in either of the treatment groups compared with that in the CTL. However, treatment of animals with 500 mg/kg FE showed a more pronounced effect. Animals in FS500, FE500 and FE1000 groups had a significantly higher level of serum TAC compared to the CTL group.ConclusionsFennel extract and seed administration in pregnancy and lactation period improve offspring’s folliculogenesis. Higher level of TAC in the serum of offspring might have positively altered the folliculogenesis milieu.

Partial luteolysis during early diestrus in cattle downregulates VEGFA expression and reduces large luteal cell and corpus luteum sizes and plasma progesterone concentration

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 μg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 μg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows.

Effect of Social Rank upon Estrus Induction and Some Reproductive Outcomes in Anestrus Goats Treated With Progesterone + eCG.

Simple SummaryThe potential effect of social rank [R] (high—HSR; medium—MSR; low—LSR) in anestrus goats subjected to an estrus induction protocol (EIP) primed with progesterone (P4) and receiving a differential equine chorionic gonadotropin (eCG) dose [D] (D100 vs D350) upon some reproductive outcomes in crossbred dairy goats under intensive stall-fed conditions was evaluated. Response variables included estrus induction (EI, %), latency to estrus (LAT, h), duration of estrus (DUR, h), ovulation (OVU, %), ovulation rate (OR, n), corpus luteum size (CLS, cm), pregnancy (PREG, %), kidding (KIDD, %), and litter size (LS, n). Most of the response variables were positively affected by social rank, favoring to the HSR goats (i.e., EI %, DUR h, OVU %, OR n, and CLS cm). In addition, increased OR and PREG occurred in the HSR + D350 group, while D350 increased LS, irrespective of R. Interestingly, since no differences regarding LAT, DUR, OVU, CLS, PREG, and KIDD occurred between D350 and D100, the obtained values support the use of a reduced level of exogenous hormones to induce and generate out-of-season reproductive efficiency.We evaluated the possible role of the social rank [R] (i.e., low—LSR, middle—MSR, or high—HSR) in anestrus goats exposed to a P4 + eCG [D] (i.e., 100 or 350 IU) estrus induction protocol (EIP). Adult, multiparous (two to three lactations), multiracial, dairy-type goats (Alpine–Saanen–Nubian x Criollo goats (n = 70; 25°51′ North) managed under stall-fed conditions were all ultrasound evaluated to confirm anestrus status while the R was determined 30 d prior to the EIP. The variables of estrus induction (EI, %), estrus latency (LAT, h), estrus duration (DUR, h), ovulation (OVU, %), ovulation rate (OR, n), corpus luteum size (CLS, cm), pregnancy (PREG, %), kidding (KIDD, %), and litter size (LS, n) as affected by R, D, and the R × D interaction, were evaluated. While OVU and CLS favored (p < 0.05) HSR (96% and + 1.04 ± 0.07 cm), an increased (p < 0.05) LS occurred in D350 vs. D100 (2.06 ± 0.2 vs. 1.36 ± 0.2); neither R nor D affected (p > 0.05; 38.5%) KIDD. However, EI, LAT, DUR, OR, and PREG were affected by the R × D interaction. The HSR group had the largest (p < 0.05) EI % and DUR h, irrespective of D. The shortest (p < 0.05) LAT occurred in D350, irrespective of R. While the largest (p < 0.05) OR occurred in HSR and MSR within D350, the HSR + D350 group had the largest PREG (p < 0.05). These research outcomes are central to defining out-of-season reproductive strategies designed to attenuate seasonal reproduction in goats.

Doppler sonography of the corpus luteum during the oestrus cycle in dairy cows

Doppler ultrasound has recently emerged as one of the main innovations in cattle practice and has mainly found application in the evaluation of luteal blood perfusion (LBF). LBF has been evaluated during different phases of the oestrous cycle, but the technique is poorly standardized. The aim of the study was to evaluate visual and quantitative changes of size of corpus luteum (CL) and LBF in dairy cattle during an oestrous cycle at 2 follicular waves using colour flow mode (CFM) and power flow mode (PFM) doppler ultrasound. Ten Friesian cows were selected and synchronized. The CL was evaluated at 3/4-day intervals until the next oestrus onset. After identifying the spiral luteal artery entry at the base of the CL, multiple scans of the vertical plane at the maximum diameter of the CL from the apex to the base were recorded in B-mode, CFM and PFM for each cow and session. An Esaote Mylab vet 30 gold was employed with the following setting: pulse repetition frequency 2.1 (CFM) or 2.8 (PFM), gain 70%. The real area of CL (RACL) was calculated subtracting the area of the eventual inner cavity. The LBF was quantified off-line by means of a visual score and of an image analysis system (Digimizer 4.1). Student and Anova tests were used for statistical analysis. Data were presented as means and standard deviations. There were significant individual variations for the RACL and LBF when calculated in CFM. PFM minimized these variations, although, at day 15-16, it was also affected by individual factors (p &lt; 0.01). The combined study of RACL and LBF during a 2-wave oestrous cycle distinguished 3 phases: a phase of CL formation (3-4 days) with LBF of 0.3 ± 0.3 cm2 on a RACL of 3.1 ± 0.9 cm2, a central phase (7-16 days) with LBF of 0.7 ± 0.4 cm2 on a RACL of 4.8 ± 0.8 cm2 and a regression phase (19-20 days) with a minimum LBF of 0.1 ± 0.1 cm2 in a CL of 3.3 ± 0.6 cm2. According to this study, there is no significant difference of LBF from 7 to 16 days of the oestrus cycle; although the RACL significantly changes in this period, the dimensional variations are too thin to be clinically appreciated. However, this study contributed to defining the values of RACL and LBF during a 2-wave oestrous cycle of dairy cattle.

105 Optimization of a five-day fixed-time embryo transfer program in dairy heifers: Use of gonadotrophin-releasing hormone at initiation of the protocol

Optimized fixed-time embryo transfer (FTET) protocols for synchronization of recipients have the potential to improve the overall efficiency and profitability of embryo transfer (ET) programs. The objective of the present study was to evaluate the effect of dose of gonadotrophin-releasing hormone (GnRH) at initiation of a 5-day synchronization protocol for FTET. Holstein heifers (n=2689) at two locations were synchronized using a 5-day CO-Synch protocol as follows: Day 0: CIDR inserted, Day 5: CIDR removed, prostaglandin (PG)F2α treatment (500μg cloprostenol), Day 6: PGF2α treatment, Day 8: GnRH (100μg of gonadorelin). On Day 0, at the time of CIDR insertion, heifers were assigned in a completely randomised design to the following groups: Single (a single dose of GnRH; 100μg of gonadorelin), Double (200μg of gonadorelin) or No GnRH (control). All heifers received an Estrotect patch placed on Day 5 and evaluated for signs of oestrus on Day 8. At location A, heifers were evaluated by ultrasonography 5 days after GnRH to determine presence and size of corpus luteum (CL), whereas at location B presence and location of CL were determined by transrectal palpation at the time of transfer. Heifers with a CL received an embryo 7±1 days after GnRH administration, and pregnancy was determined by ultrasonography 41 and 63 days after GnRH. Data were analysed by generalized linear mixed models. Oestrus expression was greater in heifers that received Single and Double GnRH than in the No GnRH group (P=0.001). Similarly, utilisation rate (number transferred per number treated) was greater for heifers in the Single and Double GnRH group than for those in the No GnRH group (P=0.02). Pregnancy data were analysed for a subset of recipients using data from Day 41 (n=2267) and Day 63 (n=2042). The analysis of fertility outcomes included as covariates the type of embryo (invitro fresh or frozen and invivo fresh or frozen), embryo stage, embryo quality, interval from GnRH to transfer, and oestrus expression. Pregnancies per embryo transfer (P/ET) at Days 41 and 63 were not different between treatment groups (P=0.86), and there was no interaction between type of embryo and treatment (P&amp;gt;0.15). Pregnancy loss between Days 41 and 63 was not different (P=0.49) between treatments groups. In conclusion, the removal of the initial GnRH from a 5-day FTET protocol resulted in a slight but significant reduction in the utilisation rate and the percentage of heifers showing oestrus. However, there was no detrimental effect on fertility. As a result, the overall cost of the FTET program can be reduced by eliminating the need for the initial GnRH treatment without compromising fertility. Table 1.Reproductive performance in recipients receiving different doses of gonadotrophin-releasing hormone (GnRH) at initiation of the synchronization protocol Treatment Oestrus (n) Utilisation rate (n) P/ET1 D41 (n) P/ET D63 (n) Pregnancy loss (n) No GnRH 69.2%B (621/898) 85.0%B (763/898) 41.6% (308/740) 39.9% (268/672) 4.3% (12/280) Single GnRH 76.1%A (685/900) 88.8%A (799/900) 42.7% (329/770) 39.5% (272/689) 6.5% (19/291) Double GnRH 75.3%A (671/891) 88.7%A (790/891) 41.5% (314/757) 38.9% (265/681) 5.4% (15/280) A,BValues with different superscripts within a column differ (P&amp;lt;0.05). 1P/ET=pregnancies per embryo transfer.

101 Treatment with gonadotrophin-releasing hormone on Day 7 or 21 does not reduce pregnancy loss in dairy heifers receiving invitro-produced embryos

Previous research has shown that induction of an accessory corpus luteum (CL) by administration of gonadotrophin-releasing hormone (GnRH) on Day 5 increases circulating progesterone and reduces pregnancy loss between Days 33 and 60 in heifers receiving invitro-produced (IVP) embryos. Therefore, the objective of the present study was to determine whether timing of induction of an accessory CL influenced pregnancy loss in IVP recipients. Holstein heifers (n=1,658) were synchronized using a modified 5-day CIDR CO-Synch protocol. Briefly, heifers received an intravaginal progesterone device (CIDR) on Day −8, Day −3: CIDR removed and first prostaglandin F2α (PGF2α) treatment, Day −2: second PGF2α, and Day 0: GnRH (G1, 100µg of gonadorelin acetate). Oestrus expression was evaluated on Day 0 with the use of an oestrus detection device. Transfer of fresh IVP embryos was performed on Day 7±1 after G1. At the time of transfer, heifers were randomly assigned, in a 2×2 factorial design, to receive GnRH (200µg of gonadorelin acetate) or remain as Control (untreated) at two different times: Day 7 (time of embryo transfer (ET)) or Day 21. Thus, the following groups were formed: Control-Control (n=410); Control-GnRH (n=409); GnRH-Control (n=419); and GnRH-GnRH (n=420). All heifers were evaluated by transrectal ultrasonography to determine number, size, and location of CL on Days 5, 14, 21, 28, 32, and 60 and pregnancy status on Days 28, 32, and 60. Data collected from each heifer included embryo stage and quality, oestrus expression, body condition score, number of transfers, and technician. Pregnancies per embryo transfer (P/ET) and pregnancy loss data were analysed by logistic regression (SAS 9.4). Ovulation to Day 7 treatment was greater (P&amp;lt;0.01) in GnRH treated heifers (70.0%; 585 out of 836) than in untreated Controls (2.7%; 22 of 819). Ovulation to Day 21 treatment was determined only in animals pregnant at Day 28 and was greater (P&amp;lt;0.01) in heifers treated with GnRH (37.6%; 153 of 407) than in untreated controls (1.0%; 4 of 390). There was no effect of Day 7 treatment (P&amp;gt;0.68) or Day 21 treatment (P&amp;gt;0.18), nor a Day 7×Day 21 treatment interaction (P&amp;gt;0.48) on P/ET at Day 32 or 60 (Table 1). Treatment with GnRH on Day 7 or 21 did not alter pregnancy loss between Days 32 and 60 (P&amp;gt;0.10). Heifers with an accessory CL present at Day 32 (11.7%; 41 of 350) had similar (P=0.55) pregnancy loss compared with heifers with no accessory CL (14.4%; 54 of 375). The number of CL present on Day 32 did not affect (P=0.23) pregnancy loss; however, heifers with 3 CL (4.9%; 3 of 61) tended (P&amp;lt;0.10) to have reduced pregnancy loss compared with heifers with 1 CL (14.4%; 54 of 375) or 2 CL (13.2%; 38 of 289). In conclusion, treatment with GnRH on Day 7 or 21 induced an accessory CL; however, it did not affect P/ET on Days 32 or 60 and pregnancy loss. Table 1.Pregnancies per embryo transfer (P/ET) and pregnancy loss in embryo recipients based on timing of treatment with gonadotrophin-releasing hormone (GnRH) Treatment P/ET Day 32% (n) P/ET Day 60% (n) Pregnancy loss% (n) Day 7 Day 21 Control Control 42.4 (410) 36.1 (410) 15.0 (174) GnRH 46.0 (409) 40.7 (409) 11.7 (188) GnRH Control 43.7 (419) 36.8 (419) 15.9 (183) P-value GnRH 43.1 (420) 38.8 (420) 10.0 (181) Day 7 treatment 0.68 0.72 0.96 Day 21 treatment 0.51 0.18 0.11 Interaction 0.48 0.66 0.71

Short communication: Accuracy of corpus luteum color flow Doppler ultrasonography to diagnose nonpregnancy in dairy cows on day 21 after insemination

The main objective of this study was to determine the accuracy of corpus luteum color flow Doppler ultrasonography (CLCFDU) to diagnose nonpregnant dairy cows 21 d after insemination. A secondary objective was to determine the accuracy of other indirect tests such as corpus luteum size and progesteronemia. Data from 1,632 Holstein cows (10 commercial herds) were used for the analysis in this prospective cohort study. The herds were visited weekly by a veterinarian and an animal health technician. During farm visits, cows were examined on d 21 after insemination using transrectal B-mode ultrasonography to quantify the presence and size of the corpus luteum. After identification of the corpus luteum, CLCFDU was performed and scored as D0, D1, D2, or D3 when 10% or less, between 11 and 30%, between 31 and 60%, or 61% or more of the corpus luteum surface was colored, respectively. A blood sample from coccygeal vessels was also collected to quantify progesteronemia. Farmers were blinded to these findings and no intervention was performed following examination. On d 32 after insemination, the cows were examined by the regular herd veterinarian using transrectal palpation and B-mode ultrasonography to determine whether the cows were pregnant or not (the reference test). Statistical analyses were conducted using 2 × 2 contingency tables. The apparent prevalence, sensitivity, specificity, positive predictive value, and negative predictive value of CLCFDU for predicting nonpregnancy were 22.0, 36.6, 99.0, 98.1, and 52.0%, respectively, when using D0 only as the diagnostic criterion; they were 47.2, 76.4, 94.8, 93.5, and 73.8%, respectively, for D0+D1 criteria. The same measures for cows with a corpus luteum <15 mm were 8.0, 11.7, 97.5, 86.9, and 43.4%, respectively, and they were 51.9, 67.4, 70.4, 76.6, and 60.0%, respectively, for progesteronemia <1 ng/mL. The measures of accuracy of CLCFDU to identify nonpregnant cows on d 21 after insemination were high, and the apparent prevalence varied depending on the diagnostic criteria used. The measures of accuracy of corpus luteum size (<15 mm) for the same purpose were high; however, apparent prevalence was low. The measures of accuracy of progesteronemia (<1 ng/mL) were low. In conclusion, CLCFDU had excellent accuracy to diagnose nonpregnancy in dairy cows on d 21 after insemination.

Effects of a single trace mineral injection at beginning of fixed-time AI treatment regimen on reproductive function and antioxidant response of grazing Nellore cows

Two experiments evaluated the effects of injectable trace minerals (ITM) administered 11 d before artificial insemination (AI) on body weight (BW), body condition score (BCS), ovarian structures, pregnancy rate, and antioxidant response of Nellore cows. In Experiment 1, 20 multiparous cows were assigned to one of two treatments: subcutaneous injection (6 mL/cow; 11 d before AI) of saline solution or ITM (60, 10, 5, and 15 mg/mL of Zn, Mn, Se and Cu, respectively) and BW, BCS, ovarian structures and blood were evaluated. In Experiment 2, 1,144 multiparous cows were assigned to same treatments described in Experiment 1 and pregnancy rate on d 30 was evaluated. In Experiment 1, ITM did not affect (P ≥ 0.23) BW, dominant follicle size, ovulation rate, and plasma concentrations of haptoglobin, ceruloplasmin and progesterone (P4). The ITM treatment tended to increase (P = 0.06) cow BCS and reduce (P ≤ 0.06) corpus luteum (CL) diameter and volume. Furthermore, ITM treatment tended to increase (P = 0.06) plasma concentrations of SOD and increased (P = 0.007) GSH-Px compared with saline injection. In Experiment 2, ITM treatment tended (P = 0.06) to increase pregnancy rate of cows with BCS ≤ 5.0 but not cows with BCS > 5.0 (P = 0.99). The ITM treatment did not alter BW, plasma P4, and acute phase response, but enhanced plasma concentrations of antioxidant enzymes, and tended to enhance BCS and pregnancy rates to AI of cows with BCS ≤ 5.0, even though there was a smaller corpus luteum size.

Relationships between size, steroidogenesis and miRNA expression of the bovine corpus luteum

In a previous study, a subset of miRNAs were identified the expression of which increases substantially during the follicle-luteal transition in cattle. Here, we investigated the functional involvement of some of these miRNAs (miR-96, miR-182, miR-132, miR-21, miR-378) by determining whether there is an association in vivo between their expression in the corpus luteum (CL), CL size and progesterone production. The two largest and two smallest CL were collected from 12 donor beef heifers on Day 7 following ovarian super-stimulation (Day 0 = 28–32 h after first standing to be mounted). Additionally, the CL and a plasma sample were collected from 29 recipient heifers on Day 15. Luteal expression of miRNAs and mRNAs, and plasma progesterone concentrations were quantified by RT-qPCR and RIA, respectively. There were no differences in the mean expression of any miRNAs examined or the steroidogenic enzymes, STAR or CYP11A1, between the largest and smallest CL in donor heifers (P > 0.1). In addition, there were no significant correlations of luteal volume or weight with any miRNA, CYP11A1 or STAR in donor heifers. However, a correlation (r ≥ 0.5, P ≤ 0.001) existed between the transcript levels of CYP11A1 and STAR in the CL, as well as between each of those and miR-182 levels. In addition, CYP11A1 abundance was moderately correlated (r ≤ 0.4, P < 0.05) with each of miR-96 and miR-378. In recipient heifers, progesterone levels were moderately correlated with luteal weight (r = 0.41, P = 0.03) but not with the expression of any miRNA, CYP11A1 or STAR (P > 0.1). Moreover, luteal CYP11A1 and STAR were correlated (r = 0.6, P ≤ 0.001) with miR-182 as well as with each other, consistent with data in donor heifers. Finally, both CYP11A1 and STAR were moderately correlated (r ≤ 0.5) with miR-132 and, in the case of STAR, with miR-378. In summary, there was no association between either luteal weight/volume or plasma progesterone concentrations and any of the miRNAs analysed in donor and recipient heifers. However, CYP11A1 and STAR transcript levels were significantly correlated with several miRNAs, most notably miR-182, as well as with each other, in luteal tissues from both donor and recipient heifers. This finding confirms results of previous in vitro studies and, importantly, provides the first in vivo evidence of a role of the miR-183-96-182 cluster in regulating luteal steroidogenesis.

Application of low dose of equine chorionic gonadotropin at acupoint Hou Hai for fixed-time artificial insemination in beef cows

This study evaluated the efficiency application of low dose of equine chorionic gonadotropin (eCG) using Hou Hai acupoint for fixed-term artificial insemination (FTAI) in beef cows. Seventy cows received intravaginal devices with progesterone, and 3 mg of estradiol benzoate on day zero (D0) of FTAI. On D9, the devices were removed, and 150 ?g of prostaglandin F2? was applied; the animals were then randomly distributed to three treatment groups: T1 (n = 23), 300 IU of eCG intramuscularly (IM); T2 (n = 23), 90 IU of eCG at acupoint Hou Hai; and T3 (n = 25), 90 IU of eCG at false acupoint IM. On D10, the animals received 1 mg of gonadotropin-releasing hormone IM, and FTAI was performed, 52 h after the devices were removed. The quantitative variables with normal distribution were assessed using analysis of variance at 5% probability. For variables that did not show normal distribution, Kruskal-Wallis test at 5% probability was used. No significant difference was noted among the groups (P &gt; 0.05) for the interval between device withdrawal at ovulation (58.2 ± 12.2 h), diameter of the largest follicle on D9 (9.9 ± 2.2 mm), diameters of the ovulatory follicle (12.2 ± 3.0 mm) and the second largest follicle (6.7 ± 2.1 mm), follicular growth rate (0.8 ± 0.3 mm/d), ovulation rate (82%), corpus luteum size (2.32 ± 0.35 cm2), and pregnancy rate (58.67%). The protocol cost per animal was US$ 10.67 (T1) and US$ 8.50 (T2 and T3). The use of 90 IU of eCG applied at Hou Hai acupoint or at false acupoint caused satisfactory synchronization of estrus in beef cows. In addition, this procedure was cost-effective.

Early pregnancy diagnosis at 21 days post artificial insemination using corpus luteum vascular perfusion compared to corpus luteum diameter and/or echogenicity in Nelore heifers

The objective was to compare the use of corpus luteum (CL) vascular perfusion to CL diameter and/or echogenicity to diagnose pregnancy at 21 d after timed-AI. Ovaries of Nelore heifers were assessed using ultrasonography in B-mode and color Doppler simultaneously 21 d after timed-AI (n = 113). Objective evaluations were performed using an image processing software to extract the number of colored pixels (ColorPix), diameter (mm) and echogenicity/mm² (EchoPix) of the CL. Subjective evaluations of the CL were performed by five evaluators using scores of estimated vascular perfusion area of color Doppler scan videos and estimated CL size and qualitative echogenicity of B-mode scan videos. The reference pregnancy diagnosis was performed 33 d after timed-AI using an ultrasonic device. Corpus luteum ColorPix, diameter and EchoPix were highly correlated (P < 0.001) with pregnancy. Pregnancy diagnosis accuracy, sensitivity, and negative predictive value were not different for CL ColorPix and diameter and was less with use of EchoPix compared to the other parameters. Size and vascular perfusion scores were correlated to the greatest extent (0.88–0.94) with the respective objective values within evaluator. The results from the ROC curve analysis indicated a smaller area under the curve for qualitative echogenicity compared to CL size and vascular perfusion. Corpus luteum vascular perfusion was the only subjective evaluation that when assessed there were no false negative pregnancy diagnoses. In conclusion, the use of the objective CL diameter resulted in the same efficacy as CL vascular perfusion evaluations for early pregnancy diagnosis in Nelore heifers.

Heat stress during the luteal phase decreases luteal size but does not affect circulating progesterone in gilts1.

Heat stress (HS) occurs when heat dissipation mechanisms are insufficient to maintain euthermia, and it is associated with seasonal infertility (SI), which manifests as smaller litters, longer wean-to-estrus interval, increased abortions, and reduced conception rates. To understand HS-induced mechanisms underlying SI, crossbred post-pubertal gilts (167 ± 10 kg; n = 14) experienced either thermal neutral (TN, 20 ± 1 °C, n = 7) or cyclical HS (35 ± 1 °C for 12 h and 31.6 °C for 12 h, n = 7) conditions from 2 to 12 d post-estrus (dpe). Estrous cycles were synchronized via altrenogest administration for 14 d, phenotypic manifestation of estrus was observed and gilts were assigned to experimental treatment. Gilts were limit fed 2.7 kg daily with ad libitum water access. Blood was collected at 0, 4, 8, and 12 dpe via jugular venipuncture and animals were humanely euthanized at 12 dpe. The corpora lutea (CL) width were measured via digital calipers on both ovaries, and CL from one ovary were excised, weighed, and protein and steroid abundance analyzed via western blotting and ELISA, respectively. Relative to TN, HS increased (P < 0.01) rectal temperature and respiration rates and reduced (P < 0.01) feed intake. The CL from HS ovaries were reduced in diameter (P < 0.05) and weight (P < 0.01) relative to those from TN animals. No difference (P = 0.38) in CL or serum progesterone concentrations between groups was observed at any time point, though at 12 dpe the serum progesterone:CL weight was increased (P < 0.10) by HS. No treatment differences (P = 0.84) in circulating insulin were observed. Luteal protein abundance of steroid acute regulatory protein, 3 beta-hydroxysteroid, or prostaglandin F2α receptor were not different between treatments (P = 0.73). Taken together, these data demonstrate that the CL mass is HS sensitive, but this phenotype does not appear to be explained by the metrics evaluated herein. Regardless, HS-induced decreased CL size may have important implications to pig SI and warrants additional attention.