Diameter Of Ovulatory Follicle Research Articles

Effect of a new device for sustained progesterone release on the progesterone concentration, ovarian follicular diameter, time of ovulation and pregnancy rate of ewes

This study evaluated the effectiveness of a new progesterone intravaginal device (DPR) in ewes through four experiments: Experiment 1 compared the circulating progesterone concentration of ovariectomized ewes that received either a new or a re-used DPR. Experiment 2 compared the progesterone concentration between DPR-estrous-synchronized ewes and naturally estrous-cycling ewes. Experiment 3 evaluated the effect of new and re-used DPRs on ovarian follicular dynamics and time of ovulation of estrous cycling ewes. Experiment 4 compared the pregnancy rate after the use of a DPR and Controlled Internal Drug Releasing Device (CIDR). The mean concentration of progesterone released by the DPR device during its first use (New Group: 5.1±0.5ng/ml) was greater than that during the second use (Re-used Group: 2.4±0.3ng/ml). There was no difference between the animals that received DPR devices for first and second use in terms of ovulatory follicle diameter, follicular wave emergence day for ovulatory follicle and period of ovulatory wave of ovarian follicular development. However, there was a significant difference between groups regarding the time between DPR device removal and first ovulation (New Group: 71.7±2.5h and Re-Used Group: 63.9±2.7h). Pregnancy rates were similar between ewes with DPR and CIDR devices. It was concluded that DPR is effective in increasing and maintaining progesterone concentrations, controlling follicular dynamics, promoting synchronized times of ovulation from healthy follicles, promoting development of a competent corpus luteum and when used results in pregnancy rates similar to that with use of the CIDR.

Effects of insecticide Thiodan®on the morphology and quantification of ovarian follicles in lambarisAstyanax bimaculatus(Linnaeus, 1758) in different treatments

Thiodan® is an insecticide which can cause morphological changes in fish tissues, dependent on the concentration. To investigate possible morphological changes, this study evaluated the number and diameter of ovarian follicles in lambaris Astyanax bimaculatus exposed to Thiodan® for 96 h in different treatments at three sub-lethal concentrations: 1.15, 2.3 and 5.6 μg L−1 and a control group without Thiodan®. Females exposed to Thiodan® were divided into four treatments: (i) without acclimation and without food, (ii) without acclimation and with food, (iii) with acclimation for 10 days and without food; (iv) with acclimation for 10 days and with food. In this study, it was noted that the action of Thiodan® at sub-lethal concentrations in a static system did not affect the morphology of follicular development. However, the treatments with acclimation showed lower numbers of primary and secondary follicles when compared to fish from treatments without acclimation. For the groups exposed to Thiodan®, a greater number of atretic follicles were observed compared to the control groups. The follicular diameter of secondary follicles in fish exposed to Thiodan® in the treatment with acclimation and with food was highest in the control group (P < 0.05). Furthermore, the mean gonadosomatic (GSI) and hepatosomatic (HSI) indices of treatments with acclimation were lower than those for treatments without acclimation. These data suggest that the fish may be being affected by the pesticide to which the fish were submitted, and may cause impairment to the follicular development.

Identification of the Expression Level to LH-r Gene In dominant and Cystic Ovarian Follicles Cells of the Cows

The present study was carried out to investigate the possible etiology and pathology of cystic ovarian follicles by determining the expression levels of mRNA for LH-r gene in follicular cells of dominant follicles in comparison with cystic ovarian follicles in Iraqi cows. This study were performed in two steps, firstly aspiration of follicular fluids from dominant follicles and cystic follicles and stored in (-20 ℃) until estradiol and progesterone assay, secondary section of the follicular wall in to two hemispheres, and stored at -70 ℃ to -80 ℃ to molecular study. The macroscopic examination of the ovaries revealed that the numbers of dominant follicles are [1] samples while cystic follicles were in [2] samples. There was a significant difference (P<0.05) in the diameter of cystic ovarian follicles (37.56 ± 0.64 mm) compared with dominant follicles (19.93 ± 0.32 mm). Results of hormonal assay showed higher estradiol-17β (865.96±10.64 ng/ml) and progesterone (84.8±1.35 ng/ml) concentrations in follicular fluids of cystic ovarian follicles, which were significantly higher (P<0.05) in comparison with those of dominant follicles which were (314.39±2.55 ng/ml) and (50.25±1.57 ng/ml) respectively. Molecular study, to evaluate the relative quantification of LH-r gene in dominant and cystic follicular cells, has been done by extraction of the total RNA and assay its concentration from these cells, synthesis data of the complementary DNA (cDNA), that done by reverse transcription PCR (q-RT-PCR) technique, of target gene and compared of the gene expression in dominant and cystic follicular cells, the our results referred to down regulation of LH-r gene expression in follicular cells of the cystic ovarian follicles, that may be assistance to understand the etiology and pathology of this case (disease), thus the up-regulation of the this gene in cells of dominant follicles may explain the important role of the LH in ovulation mechanism and increasing follicular ovulation chance.

167 THE EFFECT OF ELEVATED TEMPERATURE ON THE HEAT SHOCK PROTEIN 70 (HSP70) EXPRESSION IN BOVINE CUMULUS&amp;ndash;OOCYTE COMPLEXES AFTER IN VITRO MATURATION

Elevated temperature during in vitro maturation negatively affects the oocyte's developmental competence, leading to disturbances in nuclear and cytoplasmic maturation. In previous studies, the role of cumulus granulosa cells (CGC) in cumulus-oocyte complexes' (COC) response to elevated temperature was underestimated. However, CGC play an essential role in folliculogenesis, supporting the oocyte's metabolism as well as meiotic progression. Thus, CGC may be engaged in COC response to heat shock. Heat shock protein 70 (HSP70) is the major protein engaged in cellular response to heat stress. The aim of this study was to determine the HSP70 expression in both CGC and oocytes in response to elevated temperature after COC in vitro maturation. COC were collected from bovine ovarian follicles (diameter 2–6 mm) from slaughtered cows and divided into two treatment groups: I (control) – COC were in vitro matured in control temperature (38.5°C); II (experimental) – COC were in vitro matured in elevated temperature (41°C). After in vitro maturation they were mechanically separated into CGC and oocyte. In vitro maturation was conducted in TCM199 25 mM HEPES medium supplemented with 10% FBS, 0.02 IU mL–1 NIH-pFSH, 1 μg mL–1 17β-oestradiol, 22 μg mL–1 Na-pyruvate and 10 μg mL–1 gentamicin, adjusted to pH 7.4 in 5% CO2. The HSP70 expression in CGC and oocytes was performed by real-time PCR and normalized to S18/H2A and S18 gene expression, respectively. In addition, the immunocytofluorescent analysis of HSP70 expression in CGC and oocyte's were performed. The HSP70 was stained using primary monoclonal mouse antibodies raised against bovine HSP70 and secondary antibody raised against mouse conjugated with Alexa 488. Nuclei were stained with Hoechst 33342. Slides were analysed under laser confocal microscope (FV-500, Olympus, Center Valley, PA, USA). The HSP70 expression in CGC was measured as total optical density of HSP70 and normalized to total optical density of nuclei. Statistical analyses were performed by Portable Statgraphics 5.0 Centurion. Mean values of HSP70 expression in CGC and oocytes in real-time PCR and immunofluorescent analysis in CGC were compared using Tukey's HSD test (α = 0.01). After in vitro maturation, the expression of HSP70 in CGC was higher (0.13 ± 0.052 a.u., n = 35) in experimental temperature compared to the control (0.058 ± 0.008 a.u., n = 35) (P &lt; 0.01). In oocytes, HSP70 expression in experimental temperature (32.5 ± 5.2 a.u., n = 12) was similar to control (29.8 ± 5.6 a.u., n = 12). The immunofluorescent analysis confirmed data from real-time PCR analysis, indicating that the HSP70 expression in CGCs in experimental temperature was higher (0.46 ± 0.07 a.u., n = 10) compared to the control (0.12 ± 0.02 a.u., n = 10; P &lt; 0.01). After COC in vitro maturation in stress conditions, the HSP70 expression was up-regulated in CGC, but not in oocytes. That may indicate that CGCs play the major role in COC response to elevated temperature; however, the analysis of other heat shock proteins expression in CGC and oocytes need to be conducted. Research was supported by 505-10-023300-K00169-99.

Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year

Our hypothesis was that fertility could be increased in a timed artificial insemination (TAI) protocol based on estradiol (E2) and progesterone (P4) by combining GnRH with E2-benzoate at the start of the protocol to increase circulating P4 during preovulatory follicle development and by using 2 prostaglandin F2α (PGF) treatments at the end to decrease P4 near TAI. Lactating Holstein cows (n=1,808) were randomly assigned during the cool or hot season of the year to receive TAI (d 0) following 1 of 3 treatments: (1) control: controlled internal drug-release insert + 2mg of E2-benzoate on d −11, PGF on d −4, controlled internal drug-release insert withdrawal + 1.0mg of E2-cypionate on d −2, and TAI on d 0; (2) 2PGF: identical to control protocol with addition of a second PGF treatment on d −2; (3) GnRH: identical to 2PGF protocol with addition of a 100-μg GnRH treatment on d −11. Pregnancy diagnoses were performed on d 32 and 60 after TAI. Season had major effects on many reproductive measures, with cool season greater than hot season in percentage of cows with corpus luteum (CL) at PGF (62.9 vs. 56.2%), ovulatory follicle diameter (15.7 vs. 14.8mm), expression of estrus (86.7 vs. 79.9%), ovulation following the protocol (89.7 vs. 84.3%), and pregnancies per artificial insemination (P/AI; 45.4 vs. 21.4%). The GnRH protocol increased percentage of cows with CL (control=56.9%; 2PGF=55.8%; GnRH=70.5%) and P4 at PGF (control=3.28±0.22; 2PGF=3.35±0.22; GnRH=3.70±0.21ng/mL), compared with control and 2PGF protocols. The GnRH protocol increased P/AI at the pregnancy diagnosis at 32d [37.3% (219/595)] and 60d [31% (179/595)] after TAI, compared with control [30.0% (177/604); 25.1% (145/604)], with intermediate results with 2PGF protocol [33.2% (196/609); 28.0% (164/609)]. The positive effects of GnRH treatment on P/AI were only detected during the cool season (GnRH=50.9%; 2PGF=44.2%; control=41.0%) and not during the hot season. In addition, the effect of GnRH was only observed in cows with low P4 (<3ng/mL) at the start of the protocol and not in cows that began the protocol with high P4. Furthermore, presence of CL at PGF interacted with follicle diameter such that cows with a CL at PGF had greater P/AI if they ovulated larger rather than smaller follicles near TAI. Thus, fertility to TAI can be improved by inducing ovulation at the beginning of an E2/P4-based protocol using GnRH treatment, particularly during the cool season of the year and in cows with low P4 at the start of the protocol.

Double-Ovsynch, compared with presynch with or without GnRH, improves fertility in heat-stressed lactating dairy cows

The objective was to compare 3 timed artificial insemination (TAI) protocols in lactating dairy cows during heat stress. Multiparous Holstein cows yielding (mean ± SEM) 29.4 ± 0.3 kg of milk/d randomly were assigned to 1 of 3 TAI protocols at 34 ± 5.1 days in milk: 1) double-Ovsynch (DO; n = 486): the cows received GnRH-7d-2α-3d-GnRH and Ovsynch56 (GnRH-7d-PGF2α-56h-GnRH-16h-AI) was initiated 7 days later; 2) Presynch-GnRH-Ovsynch (PGO; n = 453): the cows received PGF2α-14d-PGF2α-2d-GnRH and Ovsynch56 was initiated 7 days later; and 3) presynch-Ovsynch (PO; n = 435): the cows received PGF2α-14d-PGF2α and Ovsynch56 was initiated 12 days later. The ovulatory response to the first GnRH of Ovsynch56 was higher in DO (65.0%) compared to PGO (53.2%) and PO (45.5%). Luteolytic response to PGF2α of Ovsynch was similar among TAI protocols (90.1%, 87.1%, and 86.2% for DO, PGO, and PO, respectively). Synchronization rate was greater in DO (86.2%) than in PGO (78.1%) and PO (72.1%) protocols. Irrespective of the TAI protocol, cows that ovulated in response to first GnRH had greater response to PGF2α (92.7 vs. 77.1%). Mean (±SEM) diameter (mm) of ovulatory follicle at TAI was larger in DO (16.1 ± 0.3) than PGO (15.6 ± 0.21) and PO (15.2 ± 0.12). Cows subjected to DO had greater P/AI at 32 days and at 60 days after TAI (26.6 and 24.4%) compared with those in PGO (21.4 and 20.0%) and PO (17.2 and 15.9%). However, TAI protocol had no significant effect on the incidence of pregnancy loss (6.1%, 6.6%, and 7.4% for DO, GO, and PO, respectively). In summary, cows in the DO protocol had a greater ovulation rate to the first GnRH and a greater synchronization rate, larger ovulatory follicles and greater P/AI. Of the 3 protocols used, DO yield the best reproductive performance in heat-stressed, lactating dairy cows.

Reproduction and diet of Hyla euphorbiacea (Anura: Hylidae) in a pine-oak forest of southeastern Puebla, Mexico

We studied some reproductive characteristics and diet of the hylid frog Hyla euphorbiacea in a pine-oak forest from Sierra Negra, southeastern Puebla, Mexico. The breeding season lasted from June to September; during this period males exhibited monthly variation in gonadal activity, whereas females did not. Sexual size dimorphism was observed, with females being larger than males, and female size was positively correlated with number and diameter of ovarian follicles. Diet during the rainy season was composed of invertebrates, classified into 16 prey categories. The most important was Lepidoptera (larvae), followed by Coleoptera (adults), and Araneae. Diet diversity and trophic niche overlap between sexes were relatively high. We discuss some recent findings and biological characteristics of H. euphorbiacea, which potentially make this species more susceptible to population declines than other members of the genus.

Pregnancy rate in lactating Bos indicus cows subjected to fixed-time artificial insemination and treated with different follicular growth inducers

The objective of this experiment was to evaluate the effects of replacing eCG with FSH or FSH/LH on the diameter of ovulatory follicles (DOF) and the pregnancy rates (PR) of lactating Bos indicus cows subjected to a synchronization of ovulation protocol and fixed-time artificial insemination (FTAI) based on progesterone, estradiol benzoate (EB), PGF2α, and estradiol cypionate (EC). On day 0, primiparous and multiparous Nellore cows (n = 329) at 68 days postpartum were body condition scored (BCS) and received a P4 implant (PRIMER 1.0 g P4) plus 2 mg EB intramuscularly (IM). At day 8, the implant was removed, and 250 μg cloprostenol were injected IM along with 0.5 mg EC. At the time of implant removal, the animals were divided into one of four treatments: cows received (G1) 1 mL of saline solution IM (Control; n = 80); (G2) 300 IU eCG IM (ECG300; n = 84); (G3) 15 mg FSH IM (Folltropin; n = 82); and (G4) 30 IU FSH/LH IM (Pluset; n = 83); all cows were subjected to FTAI 48-54 h after implant removal (day 10). Transrectal ultrasonography was performed to measure the DOF to FTAI and to estimate the pregnancy rate on day 35. A statistical analysis examined the effects of the treatment, technician, semen, BCS, calf age and parity on PR to FTAI. There was no significant difference in PR and DOF among the treatment groups (G1, 41.3%, 13.1 mm; G2, 46.4%, 14.5 mm; G3, 46.3%, 14.2 mm; and G4, 48.2%, 14.3 mm), but PR was higher in multiparous than in primiparous cows. Commercial FSH solutions (Pluset or Folltropin) do not promote increases in PR or DOF of lactating Nellore cows and are equal to eCG in promoting growth in preovulatory follicles and elevating the PR in Nellore cows with a moderate BCS subjected to a progesterone-based FTAI protocol with EC.

Asynchrony and regional differences in the reproductive cycle of the greenback stingaree Urolophus viridis from south-eastern Australia

Determining the periodicity of the reproductive cycle in chondrichthyan species when the population is recruiting asynchronously, as found for Urolopus viridis, can be problematic. The reproductive cycle generally requires distinguishable trends in reproductive indices across the population. The present study utilised other similar and sympatric urolophid species with synchronous reproductive cycles. Through data collected in the present study and comparisons of maximum total length (TL), periodicity of egg and embryo in utero, ovarian cycles, largest ovarian follicle diameter, and matrotrophic contribution (percentage increase from egg to embryo after maternal histotroph supplement) from similar studies, an annual reproductive cycle can be hypothesised. Sampling across two separate regions of Lakes Entrance (LE) and Western Bass Strait (WBS), U. viridis also showed regionality in several of the reproductive indices. Maximum TL and mass for females, mean size-at-birth, and female size-at-maturity and size-at-maternity in LE were markedly smaller than in WBS. In both regions litter size (1–2) increased with TL, with an exception of one female in WBS producing a litter of 3 which could be attributed to the larger TL. The implication of U. viridis producing such few young annually is they have the lowest biological productivity of any urolophid species in south-eastern Australia.

Ovulatory response and luteal function after eCG administration at the end of a progesterone and estradiol’ based treatment in postpartum anestrous beef cattle

The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to fixed-time AI (FTAI) on follicular dynamics, ovulation, corpus luteum (CL) development and serum progesterone concentrations. Multiparous suckled Hereford cows (n=46) in anestrus with 60–75 days postpartum were used. Females received an intravaginal device containing 0.5g of progesterone during 8 days and 2mg of estradiol benzoate i.m. at device insertion. At device removal 500μg of cloprostenol and 0.5mg of estradiol cypionate were administered i.m., and FTAI was performed 52–56h later. Cows were divided into two experimental groups to receive 400IU of eCG i.m. at device removal (n=23), while control group did not receive eCG (n=23). Daily ovarian ultrasonography (7.5MHz transducer) and progesterone concentrations determined by RIA were assayed from device removal until 30 or 14 days after FTAI, respectively. Treatment with eCG increased ovulation rate [65.2% (15/23) vs. 30.4% (7/23); P=0.018], ovulatory follicle diameter (14.5±0.4 vs. 13.1±0.7mm, mean±SEM; P=0.081), CL area from 6 to 14 days after FTAI (344.3±25.1 vs. 274.2±23.9mm2; P=0.045) and mean serum progesterone concentrations from FTAI to 14 days later (3.0±0.2 vs. 1.8±0.2ng/ml; P=0.001), in comparison with control cows. In conclusion, the addition of eCG to a progesterone and estradiol’ based treatment for FTAI improves ovulation rate and luteal function in anestrous cows. These findings have implications in order to increase pregnancy rates in FTAI treatments in Bos taurus beef cattle.

Optimizing estrous period characteristics of crossbred cows exhibiting prolonged estrus using a PGF2α analogue

The present study aimed at optimization of estrous period of prolonged estrus exhibiting crossbred cows (n=10) using PGF2α analogue (cloprostenol 500µg, i/m) administration between days 7 to 9 post-ovulation. Using a scoring chart, cows were scheduled for visual estrus detection, twice per day for 30 min. After each schedule, ultrasonography was done to detect the ovulation time. A scoring scale was used for the recording of visual estrus signs and estrus related alterations in the genital tract. During spontaneous-estrus (124.5±24h), ovulatory follicles had continuous growth before ovulation. During induced-estrus (32±3.5h), the maximum diameter of ovulatory follicle was similar to spontaneous- estrus. The corpus luteum (CL) exhibited slow regression during spontaneous-estrus. The inconsistent estrous score recorded during the extended estrous period was normalized at induced-estrus. In summary, the slow ovulatory follicle growth and CL regression could be associated with the persistence of estrous characteristics, which were optimized following PGF2α administration during early-luteal phase of cows.

Increasing length of an estradiol and progesterone timed artificial insemination protocol decreases pregnancy losses in lactating dairy cows

Our hypothesis was that increasing the length of an estradiol and progesterone (P4) timed artificial insemination (TAI) protocol would improve pregnancy per artificial insemination (P/AI). Lactating Holstein cows (n=759) yielding 31±0.30kg of milk/d with a detectable corpus luteum (CL) at d −11 were randomly assigned to receive TAI (d 0) following 1 of 2 treatments: (8d) d −10 = controlled internal drug release (CIDR) and 2.0mg of estradiol benzoate, d −3 = PGF2α(25mg of dinoprost tromethamine), d −2 = CIDR removal and 1.0mg of estradiol cypionate, d 0 = TAI; or (9d) d −11 = CIDR and estradiol benzoate, d −4 = PGF2α, d −2 CIDR removal and estradiol cypionate, d 0 TAI. Cows were considered to have their estrous cycle synchronized in response to the protocol by the absence of a CL at artificial insemination (d 0) and presence of a CL on d 7. Pregnancy diagnoses were performed on d 32 and 60. The ovulatory follicle diameter at TAI (d 0) did not differ between treatments (14.7±0.39 vs. 15.0±0.40mm for 8 and 9 d, respectively). The 9d cows tended to have greater P4 concentrations on d 7 in synchronized cows (3.14±0.18ng/mL) than the 8d cows (3.05±0.18ng/mL). Although the P/AI at d 32 [45 (175/385) vs. 43.9% (166/374) for 8d and 9d, respectively] and 60 [38.1 (150/385) vs. 40.4% (154/374) for 8d and 9d, respectively] was not different, the 9d cows had lower pregnancy losses [7.6% (12/166)] than 8d cows [14.7% (25/175)]. The cows in the 9d program were more likely to be detected in estrus [72.0% (269/374)] compared with 8d cows [62% (240/385)]. Expression of estrus improved synchronization [97.4 (489/501) vs. 81% (202/248)], P4 concentrations at d 7 (3.22±0.16 vs. 2.77±0.17ng/mL), P/AI at d 32 [51.2 (252/489) vs. 39.4% (81/202)], P/AI at d 60 [46.3 (230/489) vs. 31.1% (66/202)], and decreased pregnancy loss [9.3 (22/252) vs. 19.8% (15/81)] compared with cows that did not show estrus, respectively. Cows not detected in estrus with small (<11mm) or large follicles (>17mm) had greater pregnancy loss; however, in cows detected in estrus, no effect of follicle diameter on pregnancy loss was observed. In conclusion, increasing the length of the protocol for TAI increased the percentage of cows detected in estrus and decreased pregnancy loss.

Changes in plasma oxidative stress biomarkers in dairy cows after oestrus synchronisation with controlled internal drug release (CIDR) and prostaglandinF2α (PGF2α)

This study was designed to evaluate the plasma profiles of oxidative stress biomarkers, progesterone and ovarian follicle diameter in ovulatory versus an-ovulatory cows. Twenty cows were synchronised using controlled internal drug release (CIDR) and prostaglandinF2α (PGF2α) protocol. Plasma samples were analysed for progesterone (P4), oxidative stress (OS) biomarkers; reactive oxygen metabolites (ROMs), biological antioxidant potential (BAP), oxidative stress index (OSI = ROMs/BAP × 100), advanced oxidation protein products, ceruloplasmin and glutathione (GSH). Plasma P4 concentration was greater in ovulated cows 24 hours (h) after PGF2α treatment but lower 48 h after PGF2α treatment compared with that of an-ovulated cows at those sampling sessions (P &lt; 0.05). Ovulated cows were diagnosed with greater ovarian follicle diameter compared with that of their herd mates not diagnosed for ovulation. Significant interaction of time of PGF2α treatment and ovulation status (ovulatory versus an-ovulatory) with the plasma concentrations of OSI, BAP and GSH were observed. Ovulated cows had significantly lower BAP compared with that of an-ovulated cows (P &lt; 0.05) 9 h, 48 h, 60 h and 128 h after PGF2α treatment. Plasma concentrations of GSH were lower (P &lt; 0.05) in ovulated cows than that of an-ovulated cows 60 h and 96 h after PGF2α treatment. However, OSI was greater (P &lt; 0.05) in ovulated cows than that of an-ovulated cows 9 h, 48 h, 60 h and 128 h after PGF2α treatment. Significant associations were observed between OS status and sampling time. Oxidative stress status may have important physiological role in facilitating the ovulation process in oestrus synchronised dairy cows.

140 THE EFFECTS OF INTERVALS OF MECHANICAL VIBRATION DURING IN VITRO CULTURE ON THE DEVELOPMENT OF BOVINE EMBRYO DERIVED FROM LOW-QUALITY OOCYTES

Mechanical vibration enhances the cytoplyasmic maturation of in vitro-matured (IVM) pig oocytes (Mizobe et al. 2010 J. Reprod. Dev. 56, 285–290), as well as the development of in vitro-cultured (IVC) bovine embryos (Fujita et al. 2010 Rakuno Gakuen University Graduation thesis,1–36). In this study, the effects of intervals of mechanical vibration during in vitro culture, after IVF, on the development of embryos derived from low-quality oocytes were examined. Cumulus-oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter = 2 to 6 mm) obtained from a local abattoir. In this experiment, only grade 3 oocytes (i.e. those with one layer or partially remaining cumulus cells and normal cytoplasm) were used. Groups of 20 COC were matured in 100-μL droplets of in vitro TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. Matured COC were inseminated with 5 × 106 sperms mL–1 for 18 h. After 18 h of gamete co-culture, the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). Presumptive zygotes were cultured in vitro without mechanical vibration (control; n = 467) and with mechanical vibration for 5 s at 5 min (n = 180), 10 min (n = 180), 15 min (n = 180), and 60 min (n = 200) for 9 days. Embryo development was evaluated for cleavage and blastocyst rates, on Days 3 and 7 to 9 after IVF, respectively. The cleavage and blastocyst formation rates were analysed by the chi-squared test. Vibration at 15-min intervals increased (P &lt; 0.05) cleavage rate compared to 5 min, 60 min, and control (control: 66.2 ± 22.1%; 5 min: 49.4 ± 10.2%; 10 min: 70.0 ± 7.7%; 15 min: 86.2 ± 6.6%; and 60 min: 64.0 ± 8.5%).The highest (P &lt; 0.05) blastocyst rate among the experimental groups was found with 15-min intervals for vibration (control: 21.6 ± 9.2%; 5 min: 15.0 ± 5.3%; 10 min: 22.8 ± 1.8%; 60 min: 21.5 ± 5.0%). These results indicated that the cleavage and blastocyst formation rates of IVM-IVF-IVC bovine embryos derived from low-quality oocytes can be improved by physical stimulus during IVC. In addition, it was shown that 15-min intervals of mechanical vibration elicited the highest benefit for the development of embryos.

Pregnancy per artificial insemination in lactating dairy cows subjected to 2 different intervals from presynchronization to initiation of Ovsynch protocol

A protocol for presynchronization of ovarian status with 2 injections of PGF2α given 14d apart, with the last PGF2α injection given 12 or 14d before Ovsynch increases pregnancy per artificial insemination (P/AI) in dairy cows. We determined the efficacy of reducing the interval from the last PGF2α injection (500μg of cloprostenol) of presynchronization to initiation of Ovsynch on response to treatment and P/AI. Lactating dairy cows were assigned to an Ovsynch protocol, with the initial injection of GnRH given either 9 (PRE-9; n=135) or 12d (PRE-12; n=135) after the second PGF2α injection of presynchronization. The Ovsynch protocol consisted of 2 injections of 100μg of GnRH given 9d apart and 1 injection of PGF2α given 7d after the initial GnRH injection, and cows were subjected to timed artificial insemination (TAI; 70±3.5 DIM) approximately 16h after the second GnRH injection. Body condition score (1–5 scale) was recorded at TAI. Blood samples were taken for progesterone determination at the PGF2α injection of Ovsynch, at TAI, and at 11d after TAI. Ultrasonographic examinations were done in all cows at the second PGF2α injection of presynchronization, initial GnRH injection, PGF2α injection of Ovsynch, at TAI, and 24h after TAI for cyclicity status and ovarian responses to treatments, and at 32 and 60d after TAI for confirmation of pregnancy. Overall, 29 cows (10.7%) were determined acyclic or cystic and excluded from the study. The percentage of cows responding to initial GnRH injection (62.2 vs. 61.5%) did not differ between PRE-9 and PRE-12 but more cows in the PRE-9 group failed to respond to PGF2α treatment of Ovsynch compared with PRE-12 (22.7 vs. 10.7%). Body condition score at TAI (2.9±0.02) and mean ovulatory follicle diameter (16.4±0.2mm) were not different between treatments. Overall P/AI at 32d was reduced in PRE-9 (33.6%) compared with PRE-12 (44.3%) but pregnancy losses (5.0 vs. 3.7%) did not differ between treatments. Primiparous cows in the PRE-12 group had higher mean progesterone concentration 11d after TAI and greater P/AI 32 after TAI than primiparous cows in the PRE-9 group (6.4±0.5 vs. 4.6±0.5ng/mL and 55.8 vs. 30.0%, respectively). In conclusion, reducing the interval from the last PGF2α injection of the presynchronization treatment to initiation of Ovsynch (from 12 to 9d) did not affect ovulatory response to initial GnRH injection but reduced response to PGF2α injection of Ovsynch and P/AI at 32 and 60d after TAI. The reduction in P/AI was particularly evident in primiparous cows of the PRE-9 group.

Ovarian evaluation of Girolando (Holstein × Gir) heifers submitted to a GnRH–PGF2α–GnRH protocol in the dry or rainy seasons in the tropical savannah

The Girolando breed is used in pasture-based dairy production systems in Brazil to associate the high production of Bos taurus to the rusticity and thermal adaptation of Bos indicus. This study was designed to evaluate the physiological response to a gonadotropin-releasing hormone (GnRH)-prostaglandin F2α (PGF2α)-GnRH protocol to synchronize the ovulation in 40 Girolando heifers of a pasture-based dairy production system and its relationships with the temperature and humidity index (THI) during the dry (DS) and rainy season (RS) in the tropical savannah-Brazil's cerrado biome. Responses were characterized by follicular and corpus luteum number and diameter, ovulation (D9), and pregnancy rates after first AI. Total follicle number (8.1 ± 0.3 × 8.8 ± 0.3), D9 ovulatory follicle diameter (11.9 ± 0.4 × 10.1 ± 0.4 mm), corpus luteum diameter (8.6 ± 1.3 × 3.9 ± 1.5 mm), corpus luteum score (3.7 ± 0.8 × 1.8 ± 1.0), corpus luteum diameter after AI (9.6 ± 1.6 × 3.9 ± 1.5 mm), and corpus luteum score after AI (3.2 ± 0.4 × 0.9 ± 0.6) in DS and RS differed (P < 0.01). D9 ovulation rate was 40 % (DS) and 20 % (RS), without differences (P > 0.05). Pregnancy rate was 45 % (DS) and 11 % (RS), with differences (P < 0.01). THI differed between DS and RS (P < 0.01). THI may interfere in the follicular and luteal dynamics and in the response of Girolando heifers to the GnRH-PGF2α protocol in the tropical savannah, thus reducing the chances of pregnancy at the first artificial insemination.

Behavioural, follicular and hormonal characteristics of the oestrous cycle of Mexican Criollo cattle

The objective of the present study was to describe the behavioural, ovarian and hormonal characteristics of the oestrous cycle of Criollo cattle. Twenty-two cycling cows subjected to oestrus synchronisation were used to take blood samples every 4 h, to determine luteinising hormone (LH) until ovulation was detected by ultrasound and then every 24 h until the subsequent naturally occurring ovulation to determine progesterone concentrations. At the onset of synchronised oestrus, ovaries were examined by ultrasound every 8 h to determine dominant follicles until ovulation was detected, and daily afterwards to observe ovarian dynamics until the onset of the subsequent spontaneous behavioural oestrus. Time from oestrus to ovulation was higher in synchronised than in spontaneous oestrus (P &lt; 0.01). Time from LH surge to ovulation was 40.5 h. When two- and three-wave cycles were compared, inter-ovulatory intervals were similar, growth rate of the preovulatory follicle was lower in two-wave cycles, time from onset of ovulatory wave to ovulation was higher in cycles with two waves and time from oestrus to ovulation was higher in three-wave cycles (P &lt; 0.01). Corpus luteum volume averaged 1.3 cm3 and progesterone concentration peaked at an average 6.5 ng/mL. LH peak averaged 33.5 ng/mL during oestrus in all cows. Ovulatory follicle diameter ranged from 8 to 12 mm in either two- or three-wave cycles. Results suggested that overall times from oestrus and LH peak to ovulation are longer in Criollo cows than those in other breeds. This, along with information about growth rate, size and age of ovulatory follicles, may have implications for reviewing the optimum time for artificial insemination and how reproductive technologies are applied to Criollo cattle.

Elements of functional genital asymmetry in the cow

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

The effects of the pyrethroid insecticide, bifenthrin, on steroid hormone levels and gonadal development of steelhead (Oncorhynchus mykiss) under hypersaline conditions

The San Francisco Bay Estuary and Sacramento-San Joaquin Delta (Bay-Delta) is an important breeding and nursery ground for fish. Of particular interest are salmonids that migrate through fresh and saltwater areas polluted with various contaminants including bifenthrin, a widely used pyrethroid insecticide. Male steelhead (Oncorhynchus mykiss) exposed to bifenthrin (0.1 and 1.5μg/L) for two weeks had a lower gonadosomatic index (GSI) in freshwater but were not affected by concurrent bifenthrin exposure and saltwater acclimation. Plasma estradiol-17β (E2) levels and ovarian follicle diameter of fish exposed to bifenthrin (0.1 and 1.5μg/L) in freshwater significantly increased. Under hypersaline conditions, fish exposed to bifenthrin had significantly reduced E2 levels and smaller follicles, and unhealthy ovarian follicles were observed. Given the occurrence of bifenthrin in surface waters of the Bay Delta, understanding the impact of bifenthrin on wildlife is necessary for improving risk assessments of pyrethroids in this important ecosystem.

The effect of equine chorionic gonadotropin on follicular size, luteal volume, circulating progesterone concentrations, and pregnancy rates in anestrous beef cows treated with a novel fixed-time artificial insemination protocol

The objective was to determine the effects of eCG given on the day of, or 2 days before removal of an intravaginal progestin device, on ovarian follicle diameter, luteal volume, serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 μg im) was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly assigned to receive 400 IU im eCG on Day 6 (eCG6; N = 8) or Day 8 (eCG8; N = 8), or to not receive eCG (control; N = 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 ± 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 ± 0.2 mm) or control (8.5 ± 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N = 121) and eCG8 (N = 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 ± 857.3 mm3 and 8.1 ± 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 ± 505.1 mm3 and 4.5 ± 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates.