Ultrasound-guided Follicular Aspiration Research Articles

Effect of maternal age on ATP content and distribution of mitochondria in bovine oocytes.

Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18-20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows.

Oocyte quality in adapted Bos taurus taurus cows

The objective of the current study was to determine the performance of creole cows of the Chino Santandereano breed in the ultrasound-guided follicular aspiration technique (OPU) and the quality of oocytes recovered. A total of 15 multiparous cows were selected from a herd located in the department of Santander with tropical climate. The cows were submitted to 5 sessions of follicular aspiration with an interval of 45 days. In each aspiration session, 7.9 ± 0.7 oocytes were recovered per cow, with a recovery rate of 64.9% ± 0.5 and a rate of viable and non-viable oocytes of 64.3% ± 0.6 and 35.7% ± 0.7, respectively. A difference (P < 0.05) in oocyte quality was presented for grades I, II and III (7.7 ± 0.4, 19.8 ± 0.5 and 36.8 ± 0.5, respectively). The results suggest that Chino Santandereano cows submitted to the OPU technique have a higher percentage of grade III oocytes recovered.

Fibroids and Infertility: Transabdominal Ultrasound Guided Oocyte Retrieval in Patient with Massive Multiple Leiomyomas - Case Report

The World Health Organization (WHO) defines infertility as a disease characterized by the absence of pregnancy after 12 or more months of regular unprotected sex. Couple infertility is an increasingly present problem in today’s society. We present a case of a patient with assisted reproduction program complicated with uterus altered by numerous fibroids who obstructed routine transvaginal oocyte retrival. Routine transvaginal procedure of oocyte retrival was impossible due to the size and shape of the uterus altered by numerous fibroids. The aspiration was done by transabdominal ultrasound-guided follicular aspiration. A successful embryo transfer was performed without complications. Patient was successfully delivered at 32 weeks of gestation. Main objective of this case report is to bring to the light the challenging situations that infertility specialist face, and alternative solutions that deviate from the daily routine but bring the final desired result – birth of the healthy newborn.

Oocyte Quality, In Vitro Fertilization and Embryo Development of Alpaca Oocytes Collected by Ultrasound-Guided Follicular Aspiration or from Slaughterhouse Ovaries.

Simple SummaryWe compared the morphological quality and the in vitro developmental competence of cumulus–oocyte complexes (COCs) from live vs. slaughtered alpacas. COCs were recovered using: (i) manual aspiration in slaughterhouse ovaries (n = 15 females), or (ii) ovum pick-up (OPU) in live superstimulated alpacas (n = 13). COCs recovered were morphologically evaluated. Grade I–III COCs were in vitro matured and fertilized afterwards using fresh alpaca spermatozoa. Zygotes were in vitro cultured for seven days. The proportion of COCs recovered was similar between groups, but the mean number of COCs collected from individual ovaries was greater (p < 0.05) in slaughterhouse ovaries. A higher (p < 0.05) percentage of grades III and IV and a lower (p < 0.05) percentage of grade I COCs was obtained using OPU. Although, no significant difference in the percentage of cleavage and morula formation was observed between groups, the number of blastocysts, regarding cleavage and COCs collected, was higher (p < 0.007 and p < 0.0002, respectively) in the OPU group. However, the total number of blastocysts per female did not differ between groups. The recovery rate and morphological quality of COCs was significantly higher when follicles were manually aspirated from slaughterhouse alpaca ovaries; however, a greater developmental potential was observed in oocytes obtained from live alpaca.The morphological quality and the in vitro developmental competence of cumulus-oocyte complexes (COCs) collected from in vivo or slaughtered alpacas was compared. COCs were recovered from ovarian follicles using: (i) manual aspiration in ovaries of alpacas (n = 15) sacrificed at a local slaughterhouse, or (ii) transrectal ultrasound-guided follicular aspiration (or ovum-pick-up, OPU) in live alpacas (n = 13) 4 days after the administration of an ovarian superstimulation protocol (200 UI eCG). COCs recovered from both groups were morphologically evaluated and graded. Grade I to III COCs were in vitro matured for 26 h and in vitro fertilized afterwards for 20 h using fresh alpaca epididymal spermatozoa. Presumptive zygotes from both groups were in vitro cultured for 7 days. The proportion of COCs recovered over the total number of follicles punctured was similar between groups, but the mean number of COCs collected from individual ovaries was greater (p < 0.05) in slaughterhouse ovaries. A significantly higher (p < 0.05) percentage of low-quality COCs (grades III and IV) and a lower (p < 0.05) percentage of grade I COCs was obtained using OPU. The number of blastocysts, regarding cleavage and COCs collected, was higher (p < 0.007 and p < 0.0002 respectively) for COCs collected by OPU; however, the total number of blastocysts per female did not differ between groups. We can conclude that the recovery rate and morphological quality of COCs was significantly higher when follicles were manually aspirated from slaughterhouse alpaca ovaries; however, a statistically higher developmental potential was observed in oocytes collected by OPU from live alpaca donors.

PSXVI-16 The quality of cows’ oocyte derived at different timing regimens of transvaginal follicular puncture

Abstract Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (Ovum Pick-Up, OPU) is an essential element of modern livestock development. The aim of our work was to study the effect of two different timing regimens on the quality of oocytes, recovered from cycled Simmental heifers (n = 4). Follicle aspirations were performed once a week (1/w) or twice a week (2/w). The same donor animals were used for each timing regimens. 228 and 224 follicles were aspirated at 2/w and 1/w regimen, respectively, and 280 cumulus-oocyte complexes (COCs) were recovered (140 COCs at each of timing regimen), which corresponds to the recovery rate of 61.4±5.9% and 62.1±6.3%. On average, 4.4 oocytes were received from each donor per session. We observed a significant increase by 1.2 times (P &amp;lt; 0.05) of the proportion of OPU-oocytes of good quality, which were characterized by normal morphology, when performing the procedure in the 2/w compared to 1/w regimen (65.7±4.0% vs. 53.6±3.0% of the total number of recovered oocytes, respectively). The proportion of oocytes at the MII stage of meiosis after 24 hours of maturation was similar in both groups: 73.3±5.6% (2/w regimen) and 74.7±2.4% (1/w regimen). Proportion of oocytes with signs of apoptotic changes was also not significantly differed between the groups: 8.0±3.1 and 9.8±3.3%, respectively. We did not observe the effect of the frequency of OPU sessions on the embryo cleavage rate (on average 63.5%) and the blastocysts development rate (on average 16.7%). Our data will be used to optimize the timing regimen of oocyte retrieval from living Simmental donor heifers. The studies were performed under financial support of the RFBR (project No. 20-316-90003) and the Russian Ministry of Science and Higher Education.

Factors affecting the in vitro production of bovine embryos in a commercial program

Variability in the production of bovine blastocysts per session of ultrasound-guided follicularaspiration(OPU) is still an obstacle in commercial production.Thus, the objective of the current work was to verify factors that influence in vitro embryo production (PIVE), using data from a commercial laboratory.Data from 2014 to 2016 were analyzed, referring to 799 OPU sessions of adult bovine females of the breeds: Nelore (n=83), Girolando (n=73), Brangus (n=49), HPB (n=20), and Senepol (n=10).The influence of five variables was analyzed: breed, genetic group, frequency of aspiration, type of semen (sexed/conventional), and seasonality (spring/summer vs. autumn/winter) on the rate of viable oocytes, cleavage, and blastocyst production (bl).The protocols for the in vitroproduction of embryos (IVP) followed the routine established by the partner laboratory of the present study. The statistical analysis of the data was performed using SAS, with the Chi-square test.The Girolando and Nelore breeds had a higher (p=0.0001) number of blastocysts/OPU session, as well as the Bos indicus blood group.Blastocyst production was higher (p=0.0059) with sexed semen compared to conventional semen (6.6 x 5.2 bl/OPU session); a higher frequency of aspirations also increased (p=0.005) the number of bl/OPU session (7.7 x 5.9).We conclude that the in vitro production of bovine embryos is influenced by the analyzed factors and knowledge of these variables could guide the commercial use of OPU-PIVE.

123 Efficiency of embryo production using ovum pickup oocytes recovered from stimulated and nonstimulated cows

Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (ovum pickup, OPU) is an essential element of modern livestock development. To increase the number of OPU oocytes retrieved, hormonal stimulation is broadly applied; however, the results are ambiguous. The aim of the present study was to compare recovery rate, quality of oocytes, and efficiency of invitro production of cattle embryos using OPU oocytes derived from hormonal treated and untreated cows. The study was performed in Simmental heifers at the age of 17 to 23 months. The heifers in the first group (n=7) were previously synchronized using prostaglandin F2α (Estrumate, MSD Animal Health) and gonadotrophin-releasing hormone (GnRH; Receptal, MSD Animal Health) and then underwent superstimulation with Stimofol (Reprobiol SPRL, Belgium) in a total amount of 0.25mg of FSH and 0.05mg of LH in a treatment of 6 injections on 3 consecutive days starting on Day −4 before OPU. The heifers of the second group (n=2) underwent OPU once a week for 5 weeks (10 OPU sessions). Groups of recovered cumulus–oocyte complexes (COCs) were matured in TCM-199 containing 10% fetal calf serum, 1mM Na-pyruvate, 50μg mL−1 gentamycin, 10μg mL−1 FSH, and 10μg mL−1 LH for 24h, fertilized in Fert-TALP (Tyrode’s-albumin-lactate-pyruvate) for 18 to 20h, and cultured in CR1aa medium for 7 days. All steps of IVP were performed at 38.5°C with 5% CO2 and 90% humidity. The cleavage and blastocysts rates were assessed on Day 2 and 7, respectively. In addition, Day-7 blastocysts were fixed, and the total cell number was determined using 4′,6-diamidino-2-phenylindole (DAPI) staining. The data were analysed by ANOVA using SigmaStat software package. As expected, the number of aspirated follicles per session in the first group of cows was higher than that in the second group: 14.6±1.2 vs. 6.8±0.5 (P&amp;lt;0.05). In total, 54 and 42 COCs were collected, which corresponds to recovery rates of 54.5±7.7 and 61.3±4.9% for stimulated and nonstimulated cows, respectively. After the quality evaluation, 36 COCs of SS heifers (67.2±1.9%) and 31 COCs of non-SS heifers (75.3±5.0%) were selected for IVP. We did not observe a significant difference in cleavage rate between two groups: 77.8% in the SS group vs. 71.0% in the non-SS group, whereas the rate of blastocyst production was higher (P&amp;lt;0.05) for SS heifers (25.0%) compared with non-SS animals (16.1%). The total number of cells in Day 7 blastocysts was significantly (P&amp;lt;0.01) higher in the first group (89.8±1.3) compared with the second group (70.6±3.2). Thus, superstimulation of heifers before OPU increases the efficiency of oocyte retrieval by ultrasound-guided transvaginal follicular aspiration as well as the developmental competence of OPU-derived bovine oocytes invitro. These studies were performed under financial support of the Russian Science Foundation (project No. 19-16-00115) and the Ministry of Science and Higher Education of Russia.

Does a Large (>24 mm) Follicle Yield a Competent Oocyte/Embryo?

Aim: To evaluate the effect of large follicular size (≥24 mm) at day of oocyte retrieval on oocyte/embryo quality. Patients and Methods: A cohort study was conducted in a single tertiary medical center between July 2018 and May 2019. Before ultrasound-guided follicular aspiration, follicles were measured and divided into 2 groups according to their maximal dimensional size: large: ≥24 mm and normal: <24 mm. Microscopic examination of the follicular aspirates was performed by an embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization, and embryo quality. Results: 428 follicles were measured, including 383 (62.81%) in the normal and 45 (14.06%) in the large follicle groups. Oocytes were achieved during aspiration from 297 (75.5%) and 29 (64.4%) of the normal and large follicle groups, respectively (p = 0.05). No in-between group differences were observed in mature oocyte (MII), fertilization, and top-quality embryo (TQE) rates. Nevertheless, once a zygote (2PN) was achieved, a trend toward a higher TQE rate/2PN was found in the large follicle group (16/19 [84.2%] vs. 115/171 [67.3%]; p = 0.062). Conclusion: While a nonsignificant decrease in oocyte recovery rate was found in follicles ≥24 mm, the zygote and TQE per follicle were comparable.

Do Follicles of Obese Patients Yield Competent Oocytes/Embryos?

Aim: This study evaluated the competency of oocytes/embryos derived from follicles >15 mm in diameter from obese patients, compared with nonobese patients. Patients and Methods: A cohort study was conducted in a single tertiary medical center between July 2018 and May 2019. Before ultrasound-guided follicular aspiration, follicles were measured and those with maximal dimensional size >15 mm were tracked. Microscopic examination of the follicular aspirates was performed by an embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization, and embryo quality. Results: 457 follicles were measured: 380 (83.2%) in nonobese and 77 (16.8%) in obese patients. No in-between group differences were observed in the causes of infertility, patients’ demographics, or ovarian stimulation characteristics. Oocytes were achieved during aspiration from 277 (72.8%) and 54 (70.0%) of the nonobese and obese groups, respectively (p = 0.67). No in-between group differences were observed in fertilization (2PN/oocyte), top quality embryo (TQE) per zygote (2PN), and TQE per follicle. Conclusion: Oocyte recovery rate from follicles >15 mm is unrelated to patients’ BMI. Moreover, the oocytes recovered from obese patients are competent yielding comparable zygote and TQE per follicle/oocyte, compared with nonobese patients. Further investigation is required to strengthen this finding.

The association between follicle size and oocyte development as a function of final follicular maturation triggering.

To study the association between follicle size and oocyte/embryo quality, as a function of different triggering modes for final follicular maturation. Cohort study conducted in a single tertiary medical centre between July 2018 and May 2019. All women undergoing ovarian stimulation with triggering using human chorionic gonadotrophin (HCG), gonadotrophin-releasing hormone (GnRH) agonist or dual trigger (GnRHa + HCG) were included. Before ultrasound-guided follicular aspiration, follicles were measured and divided into three groups according to maximum dimensions: large ≥16 mm, medium 13-15 mm and small <13 mm. Microscopic examination of the follicular aspirates was performed by an embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization and embryo quality. A total of 640 follicles were measured, including 402 (62.8%) in the large, 148 (23.1%) in the medium and 90 (14.1%) in the small groups. Oocytes were obtained during aspiration from 76.3%, 70.3% and 55.6% of the large, medium and small follicle groups, respectively (P = 0.001). The mature oocyte (metaphase II) rate was significantly higher in the large (P = 0.001) and medium (P = 0.01) compared with the small follicle group. Nevertheless, no between-group differences were observed in fertilization or top quality embryo rates among mature oocytes regardless of the size of the follicle from which they originated. Triggering mode did not influence oocyte recovery rate in the different follicle size groups. A higher oocyte recovery rate was observed from follicles >13 mm, however, mature oocytes achieved similar fertilization and top quality embryo rates regardless of follicle size. Triggering mode did not influence oocyte recovery rate.

Influence of temperature-humidity index on conception rate of Nelore embryos produced in vitro in northern Brazil.

Considering the high temperatures that the tropical climate provides to most of Brazil and the effects of thermal stress on reproductive processes, the objective of the present study was to analyze, in the warmer months of 2016, conception rates of Nelore bovine embryos in Acre state. For this purpose, oocytes were aspirated (ultrasound-guided follicular aspiration), matured, fertilized with Nelore bull semen, cultured for 6 days, and then the embryos were transferred to crossbred recipients. Pregnancy diagnosis was performed 30 and 60 days after embryo transfer. Meteorological data were obtained at www.inmet.gov.br to generate temperature-humidity index (THI). The data from the conception rates and periods of the year were submitted to the chi-square test at 5% probability to verify independence. Regression analysis was used to verify the relationship between THI and gestation rate. There was a strong relationship between conception rates and THI values, verified by an increase in conception rates as THI values were reduced and a decrease when THI reached the highest value. Our findings demonstrated a negative effect of heat stress in conception rates of crossbred cows in northern Brazil.

First attempts for vitrification of immature oocytes in donkey (Equus asinus): Comparison of two vitrification methods

Most wild donkey breeds are severely threatened by poaching for meat, habitat loss, and competition with livestock for food resources. Moreover, due to the mechanization in agriculture and in transport, most domestic donkey breeds are at risk of extinction. Considering the importance of biodiversity and preservation of genetic resources, the creation of genetic banks for endangered donkey breeds is urgently needed. Cryopreservation of immature jennies oocytes would be an efficient tool to allow storage of female genetics. The aim of the present study was to establish conditions for immature donkey oocyte vitrification, using equine oocytes as a control. Asine and equine immature cumulus-oocyte complexes were collected by transvaginal ultrasound-guided follicular aspiration and flushed to obtain oocytes surrounded by only corona radiata. Oocytes were vitrified after exposure to increasing concentrations of dimethyl sulfoxide, ethylene glycol and sucrose as cryoprotectants in a solution of INRA-Freeze™ medium or TCM199-Hepes supplemented with bovine serum albumin. Oocytes were warmed in decreasing concentrations of sucrose and processed for in vitro maturation. The recovery rate was 48% for jennies oocytes (4.8 oocyte per female) and 42% for mares oocytes (3.5 oocyte per female). When oocytes were exposed to cryoprotectants in INRA-Freeze™ medium none of the jennies re-warmed oocytes matured, whereas 24% of the mares re-warmed oocytes reached metaphase II after in vitro maturation. When oocytes were exposed to cryoprotectants in TCM199-Hepes-BSA medium, 33% of the jennies re-warmed oocytes matured. In conclusion, we developed a method for the vitrification of immature oocytes from jennies that allows in vitro maturation of the vitrified-warmed asine oocytes. Their competence for fertilization and development has to be ascertain.

Differential expression of LHCGR and its isoforms is associated to the variability in superovulation responses of Gir cattle

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ± 1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ± 0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (−0.5 ± 0.2 and −0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.

167 Basal and maximal oxygen consumption of oocytes from young and old mares

Mitochondria play a critical role in oocyte developmental competence by providing energy to the oocyte through oxidative phosphorylation. We hypothesised that oxygen consumption, a measure of mitochondrial function, would be lower in the oocytes of old versus young mares as a potential mechanism of reduced developmental competence associated with equine aging. To test this hypothesis, we used a novel electrochemical sensor device to compare basal and maximal oxygen consumption rates of oocytes obtained from mares aged 6 to 12 years (Young, n=8 from 7 mares) and=20 years (Old, n=12 from 8 mares). Cumulus-oocyte complexes (COC) were collected by transvaginal, ultrasound-guided follicular aspirations of dominant follicles (35mm) during oestrus and at 16±2h after induction of follicular maturation (2000IU of hCG and 0.75mg of deslorelin acetate). Recovered COC were incubated in medium [TCM-199; Life Technologies, Grand Island, NY, USA) with 10% fetal calf serum, 25mg mL−1 gentamicin, and 0.2mM pyruvate] at 38.5°C in 5% CO2 and air for 26±2h. After maturation, COC were stripped of cumulus cells and checked for the presence of an extruded polar body before electrochemical measurements using amperometry. A microchamber with a 3-electrode system (working, counter, and reference electrodes) was filled with 120µL of medium (G-MOPS™, Vitrolife, Englewood, CO, USA) and overlaid with 120µL of paraffin oil (OVOIL™, Vitrolife). The baseline current was measured before adding the oocyte through the oil layer and over the centre of the working electrode. Oxygen consumption was monitored as the linear decrease in the oxygen reduction current over time, which was converted to femtomoles of O2 consumed per second (fmol s−1) using a calibration curve for the sensor. Basal (nonstimulated) oxygen consumption was measured for 5min, followed by assessment of maximal noncoupled respiratory capacity obtained by titrating 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 8-min intervals. The highest value observed during CCCP titrations was considered the maximal oxygen consumption. Two-tailed Student’s t-tests were used to analyse data. Oocytes from young mares had higher basal oxygen consumption compared with old mares, respectively (mean±s.e.m., 1.74±0.18 and 1.27±0.09 fmol s−1; P=0.04) as well as higher maximal oxygen consumption (2.67±0.19 and 2.05±0.12 fmol s−1; P=0.016). Mitochondrial efficiency (maximal/basal oxygen consumption) was not different for Young and Old, respectively (1.62±0.13 and 1.65±0.07; P=0.9). In summary, we used a novel electrochemical sensor device to measure basal and maximal oxygen consumption of oocytes and to confirm our hypothesis that aging impairs mitochondrial respiratory capacity in equine oocytes, which may contribute to age-associated changes in equine fertility.

Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1)

ABSTRACT Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P&lt; 0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.

4 Relationship Between Ovarian Vascularity, Cumulus–Oocyte Morphology and Luteal Development in Four-Month-Old Calves After FSH Stimulation

The objectives of this study were to determine the effect of LH on the blood flow to the ovaries of 4-month-old calves after 2 FSH stimulation protocols, and to examine the relationship between ovarian vascularity after superstimulation to the morphology of the cumulus–oocyte complexes (COC) and luteal function. We hypothesise that ovarian vascularity (detected by 3-dimensional (3D) analysis of Doppler ultrasound cineloops) will increase in response to LH, and the magnitude of change in vascularity would be predictive of (1) a greater proportion of expanded COC, (2) greater development of luteal tissue volume and vascularity at 3 and 7 days after follicle aspiration, and (3) higher levels of plasma progesterone. Ovarian superstimulation was initiated at the beginning of an induced follicular wave in 4-month-old beef calves (n = 16), and beef cattle &gt;16 months of age (control group, adults; n = 8) using either a traditional 4-day or an extended 7-day FSH protocol (n = 8 calves and n = 4 controls per group). Power Doppler ultrasound cineloops were recorded immediately before (i.e. 12 h after the last FSH treatment) and 24 h after LH treatment (before ultrasound-guided follicular aspiration for oocyte collection) to assess ovarian vascularity, and 3 and 7 days after follicular aspiration to assess luteal tissue volume and vascularity. Video segments were analysed in Fiji and Imaris software to obtain the 3D ovarian vascularity index (ratio of blood flow volume to tissue volume). The ovarian vascularity index tended to increase &gt;1.7-fold in response to exogenous LH in both prepubertal calves (pre-LH 1.5 ± 0.4% v. post-LH 2.6 ± 0.7%; P = 0.08) and adult cattle (pre-LH 2.2 ± 0.6% v. post-LH 4.7 ± 0.9%; P = 0.07). Calves with a recovery of &gt;75% of expanded COC had a higher ovarian vascularity index (10.7 ± 2.6% v. 4.8 ± 1.6%; P = 0.06) and luteal vascularity index (15.7 ± 4.5% v. 5.7 ± 2.1%; P &lt; 0.05) 7 days after aspiration than those with &lt;75% expanded COC. Calves in the 7-day FSH protocol had &gt;10-fold higher concentration of plasma progesterone on Day 3 (12.7 ± 7.3 ng mL−1 v. 1.2 ± 0.4 ng mL−1; P &lt; 0.05) and Day 5 (50.6 ± 28.0 ng mL−1 v. 4.5 ± 1.0 ng mL−1; P &lt; 0.05), and ~2-fold higher luteal vascularity index at 7 days after follicle aspiration (13.7 ± 4.6% v. 7.7 ± 2.8%; P &lt; 0.05) than calves in the 4-day FSH protocol, whereas no difference (P &gt; 0.05) was found in control (adult) animals. In conclusion, there was an increase in ovarian vascularity resulting from LH treatment in prepubertal calves and adult cattle. A greater proportion of expansion of COC at 24 h after LH treatment (an indicator of follicular maturation) was related to higher ovarian and luteal vascularity on Day 7 after collection in prepubertal calves, but not in adults. Luteal vascularity on Day 3 was reflective of plasma progesterone concentration, and prepubertal calves in the 7-day FSH protocol had greater plasma progesterone than calves in the 4-day FSH protocol. The use of FSH in calves allows a greater number of follicles for oocyte collection as it does in adult cattle. Research was supported by an NSERC grant.

A 100-Year Review: Reproductive technologies in dairy science

Reproductive technology revolutionized dairy production during the past century. Artificial insemination was first successfully applied to cattle in the early 1900s. The next major developments involved semen extenders, invention of the electroejaculator, progeny testing, addition of antibiotics to semen during the 1930s and 1940s, and the major discovery of sperm cryopreservation with glycerol in 1949. The 1950s and 1960s were particularly productive with the development of protocols for the superovulation of cattle with both pregnant mare serum gonadotrophin/equine chorionic gonadotrophin and FSH, the first successful bovine embryo transfer, the discovery of sperm capacitation, the birth of rabbits after in vitro fertilization, and the development of insulated liquid nitrogen tanks. Improved semen extenders and the replacement of glass ampules with plastic semen straws followed. Some of the most noteworthy developments in the 1970s included the initial successes with in vitro culture of embryos, calves born after chromosomal sexing as embryos, embryo splitting resulting in the birth of twins, and development of computer-assisted semen analysis. The 1980s brought flow cytometric separation of X- and Y-bearing sperm, in vitro fertilization leading to the birth of live calves, clones produced by nuclear transfer from embryonic cells, and ovum pick-up via ultrasound-guided follicular aspiration. The 20th century ended with the birth of calves produced from AI with sexed semen, sheep and cattle clones produced by nuclear transfer from adult somatic cell nuclei, and the birth of transgenic cloned calves. The 21st century has seen the introduction of perhaps the most powerful biotechnology since the development of artificial insemination and cryopreservation. Quick, inexpensive genomic analysis via the use of single nucleotide polymorphism genotyping chips is revolutionizing the cattle breeding industry. Now, with the introduction of genome editing technology, the changes are becoming almost too rapid to fully digest.

Transabdominal Ultrasound-guided Follicular Aspiration in Case of Müllerian Anomaly with Ovarian Malposition

ABSTRACT Objectives: To describe the approach of transabdominal ultrasound-guided oocyte retrieval in a patient of Müllerian anomaly and ovarian malposition. Design: Case report. Setting A tertiary referral reproductive medicine unit. Patient A 23-year-old patient with Müllerian anomaly (right unicornuate with left rudimentary horn uterus) and ovarian malposition. Intervention Controlled ovarian hyperstimulation, transabdominal ultrasound-guided oocyte retrieval. Main outcome measure Total and mature oocytes retrieved, fertilization rate, embryo number, and quality. Results Five oocytes were retrieved transabdominally, using percutaneous needle puncture under ultrasound guidance. Conclusion In in vitro fertilization protocols, patients with Müllerian anomaly with vaginally inaccessible ovaries, transabdominal ultrasound-guided follicular aspiration is a safe and efficacious alternative technique for oocyte retrieval. How to cite this article Mishra VV, Choudhary S, Goyal PA, Aggarwal R. Transabdominal Ultrasound-guided Follicular Aspiration in Case of Müllerian Anomaly with Ovarian Malposition. J South Asian Feder Obst Gynae 2017;9(1):53-55.

Changes in ovarian function associated with circulating concentrations of estradiol before a GnRH-induced ovulation in beef cows

These studies were conducted to evaluate causes for differences in circulating concentrations of estradiol before a GnRH-induced ovulation. Beef cows were synchronized by an injection of GnRH on day −7 and an injection of prostaglandin F2α (PGF2α) on day 0. In experiment 1, blood samples were collected every 3 h from PGF2α on day 0 to hour 33 after PGF2α and at slaughter (hour 36 to 42; n = 10). Cows were assigned to treatment group based on circulating concentrations of estradiol (E2): HighE2 vs LowE2. At slaughter, follicular fluid (FF) and granulosa cells were collected from the dominant follicle. In experiment 2, blood samples (n = 30) were collected every 8 h from PGF2α until the dominant follicle was aspirated via ultrasound-guided follicular aspiration to collect FF and granulosa cells (hour 38 to 46). In experiment 1, HighE2 had increased abundance of 3β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.02), and greater concentrations of estradiol and androstenedione (P ≤ 0.02) in the FF. In experiment 2, HighE2 had increased abundance of CYP11A1, 3β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.03) vs either LowE2 or GnRHLowE2. There was a tendency (P = 0.07) for LH pulse frequency to be increased in both the GnRHLowE2 and HighE2 compared with LowE2. HighE2 cows experienced increas in circulating concentrations of estradiol compared with LowE2. In conclusion, animals with greater concentrations of circulating estradiol before fixed-time AI experienced an upregulation of the steroidogenic pathway during the preovulatory period.

Comparing transabdominal and transvaginal ultrasound-guided follicular aspiration: A risk assessment formula

ObjectiveWe sought to identify patients at risk of incomplete transvaginal oocyte retrieval, develop a risk assessment formula to identify patients who would benefit from a transabdominal approach, and compare complication and pregnancy rates between these two approaches. Materials and MethodsIn this retrospective case control study in a private in vitro fertilization center, 95 cases of women undergoing transabdominal follicular aspiration for oocyte retrieval (15 transabdominal only and 80 transabdominal and vaginal combined) were compared with 278 controls of women undergoing the transvaginal aspiration only. Transabdominal oocyte retrieval was performed when one or more ovaries could not be retrieved via the transvaginal approach. Main study outcomes included need for transabdominal retrieval, pregnancy rates, and complications. ResultsA risk assessment scoring system was developed as follows: difficulty seeing ovaries on ultrasound (+4), history of pelvic surgery (+3), and body mass index of 30 kg/m2 or greater (+2). With a cutoff score of 4 or greater, the overall sensitivity is 75%, specificity is 80%, positive predictive value is 57%, and negative predictive value is 90%. No statistically significant differences were found for pregnancy rates or complications. ConclusionThe transabdominal approach is an alternative option that would increase the total number of oocytes retrieved with no statistical difference in complication or pregnancy rates. We also developed a scoring system that can serve as a useful screening tool for identifying women at increased risk of transabdominal oocyte retrieval.