Transvaginal Follicular Aspiration Research Articles

Prolonged follicular dominance is associated with dysregulated proteomic profile of the follicular fluid in Bos indicus cows

Prolonged follicular dominance is one of the conditions associated with disconcerted follicular dynamics that result in substantial economic losses to the farmers through low reproductive efficiency in cattle. Hormonal aberrations associated with prolonged follicular dominance may affect the follicular microenvironment and composition of follicular fluid. The current study focused on proteome changes of follicular fluid in prolonged follicular dominance compared to physiological follicular dominance. Prolonged dominance was induced in Deoni cows (n = 6) by using CIDR (previously used for 7 days) from day 4–8 of estrus, with PGF2 injection on day 6 and day 7 at 12 h intervals. Follicular fluid was collected by ultrasound guided transvaginal follicular aspiration method. Global proteomic analysis of follicular fluid revealed 217 proteins in the Deoni cow, with the majority of proteins involved in 21 pathways, 42 molecular functions, and 106 biological processes. Complement and coagulation cascades (22.8%) and cholesterol metabolism (4.68%) were the major pathways in which identified proteins were involved. Comparison of physiological and prolonged dominant follicular fluid revealed differential expression of 26 proteins, of which 15 were upregulated and 11 were downregulated. Proteins involved in complement and coagulation cascades, and vitamin digestion and absorption were found to be dysregulated in PFD. The present study suggests that the expression of proteins involved in inflammation, oocyte metabolism, and ovulation cascade were found to be dysregulated in the follicular fluid of prolonged follicular dominance consequently resulting in delayed ovulation or anovulation.

Effect of cooled vs frozen stallion semen on the efficiency of equine in vitro embryo production

Since 2008, our practice has been optimizing the equine in vitro embryo production system. Oocyte collection, in vitro maturation and culture media, sperm processing techniques and the Piezo driven sperm injection have remained relatively unchanged since then. However, our population of patients is now younger donor mares (< 17 years old) and in some instances, the use of extended-cooled semen has been prioritized. The purpose of this clinical report is to provide evidence that utilization of cooled semen 24-36 h post collection for ICSI increases the overall embryo production efficiency. Mares (n=350, 90% <17 years old and 8 different breeds) were presented for transvaginal follicular aspiration (TVA) and ICSI. Antral follicle count was determined prior to TVA and oocyte retrievals (n=680) were performed between day 12-20 post ovulation, or 19-25 days after the last TVA. In vitro embryo production was performed as previously described (LF Campos-Chillon, CM Owen, JL Altermatt, J Equine Vet Sci. 2019; 73:51-55). Briefly, sperm injections were performed with a Piezo driven system with the client's stallion of choice (n=262), and in vitro embryo culture was done for up to 9 days. Data (frozen vs cooled semen) was analyzed by One-way ANOVA using Minitab. The average follicle count was 15 (2-35) with an oocyte recovery rate of 82 ± 8%. Oocyte maturation rate expressed as oocytes witha polar body was 70.6 ± 1.1%; for frozen (n=476) vs cooled (n=204) semen injections, cleavage (57.6 ± 1.8 and 70.1 ± 2.9%) and blastocyst (13.4 ± 1.2 and 25.8 ± 1.5%) rates were different (p<0.001) respectively. In addition, number of embryos produced per cycle were 1.16 ± 0.08 for frozen and 2.24 ± 0.13 for cooled semen (p<0.001). Pregnancy rates of fresh embryo transfers at day 35 were higher (p<0.004) for embryos obtained with cooled (n=90, 91.5 ± 0.04%) than with frozen (n=250, 72 ± 0.05%) semen. Furthermore, of the 23% of cycles (n=156) that did not produce an embryo in the first TVA attempt, 18 and 82 % were performed with cooled and frozen semen, respectively. Overall, when cooled semen was used, there was a one-fold increase in embryos obtained per TVA, 20 percentile points higher pregnancy rate, and only 4 % of cycles using cooled semen yielded no embryo vs 19% with frozen semen. These results suggest that cooled semen used for ICSI increases the overall efficiency of the in vitro embryo production system. However, further prospective studies controlling stallion and mare effect of cooled and frozen semen in the model are warranted. In addition, studies involving sperm motility/viability, decondensation parameters and PLCz content and its effect in semen samples as recently described by Gonzales-Castro (Repro Fert Dev. 2022, 35:150-150).

Effect of melatonin in maturation medium on equine oocyte maturation, cleavage, and blastocyst production following ICSI

Melatonin has been shown to have beneficial effects in in vitro oocyte maturation and embryo production through antioxidant activity but also through melatonin receptors on oocytes and cumulus cells as well. The purpose of this study was to investigate the effects of melatonin in maturation medium on equine oocyte maturation, cleavage, and blastocyst production with maturation performed in a reduced oxygen atmosphere. Oocytes were recovered via trans-vaginal follicular aspiration and divided randomly into three groups each with 5 replicates. The oocytes were placed in maturation medium containing no added melatonin (Control), 10−9 M melatonin (Group 1), or 10−7 M melatonin (Group 2) in an atmosphere of 5% CO2, 5% O2, and 90% N2. ICSI was performed on metaphase II oocytes after 28-30 hours of maturation. Presumptive zygotes were observed on day 2 post-injection for cleavageand on days 5 through 10 for blastocyst development. Results: No significant difference (p>.01) was determined for maturation to metaphase II, 59/86 (68.6%), 63/88 (71.5%), 66/89 (74.2%) for Control Group, Group 1, and Group 2 respectively. No significant difference (p>.01) was determined for cleavage 43/59 (72.9%), 43/63 (68.2%), 44/66 (66.7%) for Control Group, Group 1, and Group 2 respectively. No significant difference (p>.01) was determined for blastocyst formation 19/59 (32.2%), 25/63 (39.6%), 26/66 (39.4%) for Control Group, Group 1, and Group 2 respectively. While there was a slight trend towards an increase in blastocyst production with the addition of melatonin to maturation medium under reduced oxygen concentration, the difference was not statistically significant and further work would be required to evaluate its effect.

Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal (‘coasting’). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated (‘coasted’) cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38 g P4] or PRID® Delta [1.55 g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F2α) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development.

​Effect of Season on Quantity and Competence of Oocytes Recovered Transvaginally from Holstein Cows for In vitro Fertilization

Background: Season of the year can affect the reproductive behavior in Holstein cows, altering the competition of the oocytes, reflecting a reduced production of embryos. The objective of this study was to evaluate the average of total oocytes, competition of oocytes and embryos in the in vitro production process at different season of the year in Holstein cows. Methods: During the four seasons of the year, was performed on each of the oocyte donor cows (winter, n = 957; spring, n = 1571; summer, n = 1776; autumn, n = 1128), by in vivo transvaginal follicular aspiration technique after the collection were subjected to the embryos production in vitro. Result: The highest number of total embryos were produced in winter and autumn, compared to spring and summer (3.76±0.16 and 3.54±0.18 vs. 2.73±0.11 and 2.45±0.10; respectively, P less than 0.05). During winter, a higher percentage of oocyte competition was observed, followed by autumn and spring and less competition shown in summer (26.03±0.39, 19.08±0.29, respectively, P less than 0.05). The quantity and competence of the oocytes collected and in vitro embryo production were drastically reduced during the hottest months of the year in this area of intense heat.

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.

Effects of pretreating in vitro matured of native Thai cattle oocyte with docetaxel before vitrification on their viability

The present study was to investigate the efficacy of docetaxel (DT) at reducing microtubule damage during vitrification and maintaining the developmental competence of vitrified-warmed oocytes in native Thai cattle. Native Thai cattle cow oocyte from ultrasound-guided transvaginal follicular aspiration (UTFA) were subjected to in vitro maturation (IVM). Then, the IVM oocytes were pretreating with DT in various concentration (0.0, 1.0, 2.0 and 3.0 µM) and subjected to cryopreservation by Cryotop vitrification method. The survival rate of vitrified-warmed oocyte, cleavage and blastocyst formation rate after in vitro fertilization (IVF), and blastocyst nucleic cell numbers were evaluated in this study. Pretreatment of IVM native Thai cattle oocytes with 0.05 µM DT before vitrification resulted in significantly higher (P < 0.05) rates of oocyte survival and cleavage after IVF, and subsequent blastocyst formation on Days 7-8, hatching and hatched on Days 8-9, compared with oocytes pretreated with 0.5 and 1.0 µM DT before vitrification or those vitrified and the control group. Pretreatment of IVM native Thai cattle oocytes with 0.05, 0.5 and 1.0 µM DT before vitrification without side effects on blastocyst nucleic cell numbers. In summary, pretreatment of IVM native Thai cattle oocytes with 0.05 μM DT before vitrification improved survival of vitrified-warmed oocytes, fertilization and developmental competence.

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.

Analysis of bovine blastocysts indicates ovarian stimulation does not induce chromosome errors, nor discordance between inner-cell mass and trophectoderm lineages

Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology125: 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P < 0.001) for stimulated than non-stimulated cycles (0.712 ± 0.0288 vs. 0.466 ± 0.0360), but the overall proportion aneuploidy was similar for both groups (0.241 ± 0.0231). Most abnormalities consisted of meiotic trisomies. Twenty in vivo derived blastocysts recovered from the same donors were all euploid, thus indicating that 24 h of maturation is primarily responsible for aneuploidy induction. Chromosomal errors in OPU-IVP blastocysts decreased (P < 0.001) proportionately as stage/grade improved (from 0.373 for expanded Grade 2 to 0.128 for hatching Grade 1 blastocysts). Importantly, there was a high degree of concordance in the incidence of aneuploidy between TE and ICM lineages. Proportionately, 0.94 were “perfectly concordant” (i.e. identical result in both); 0.01 were imperfectly concordant (differing abnormalities detected); 0.05 were discordant; of which 0.03 detected a potentially lethal TE abnormality (false positives), leaving only 0.02 false negatives. These data support the use of TE biopsies for PGT-A in embryos undergoing genomic evaluation in cattle breeding. Finally, we report chromosome-specific errors and a high degree of variability in the incidence of aneuploidy between donors, suggesting a genetic contribution that merits further investigation.

Recovery of Equine Oocytes in Ambulatory Practice and Potential Complications

Field collection of oocytes in mares using transvaginal follicular aspiration (TVA) for embryo production has the potential to revolutionate the equine industry. Protocols for TVA in specialized laboratory settings have been described in the scientific literature since the early 1980s. The objective of this study was to determine the success rate of TVA oocytes recovery under ambulatory conditions. A secondary goal of this study was to determine if TVA is associated with any health complications when performed by recently trained practitioners in the field. Follicles (n = 296) from 66 adult clinically healthy mares were aspirated over a period of 6 days. TVAs were performed by 22 veterinarians with 5–20 years of experience in equine and bovine reproductive medicine, but no previous experience in TVA. Oocytes (n = 145) were recovered. No short- or long-term systemic or local complications were observed following TVA in any of the mares used in this study. Fifty-six out of 66 mares became pregnant within 3 months following TVA. This study shows that with proper training, TVA can be successfully used to obtain equine oocytes with no health complications under field conditions in nonspecialized laboratory settings.

Assessing equine embryo developmental competency by time-lapse image analysis.

The timing of early mitotic events during preimplantation embryo development is important for subsequent embryogenesis in many mammalian species, including mouse and human, but, to date, no study has closely examined mitotic timing in equine embryos from oocytes obtained by ovum pick-up. Here, cumulus-oocyte complexes were collected by transvaginal follicular aspiration, matured invitro and fertilised via intracytoplasmic sperm injection. Each fertilised oocyte was cultured up to the blastocyst stage and monitored by time-lapse imaging for the measurement of cell cycle intervals and identification of morphological criteria indicative of developmental potential. Of the 56 fertilised oocytes, 35 initiated mitosis and 11 progressed to the blastocyst stage. Analysis of the first three mitotic divisions in embryos that formed blastocysts determined that typical blastocyst timing (median±IQR) is 30.0±17.5min, 8.8±1.7h and 0.6±1.4h respectively. Frequent cellular fragmentation, multipolar divisions and blastomere exclusion suggested that equine embryos likely contend with a high incidence of chromosomal missegregation. Indeed, chromosome-containing micronuclei and multinuclei with extensive DNA damage were observed throughout preimplantation embryogenesis. This indicates that time-lapse image analysis may be used as a non-invasive method to assess equine embryo quality in future studies.

In vitro embryo production in buffaloes: from the laboratory to the farm.

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.

Alteraciones macroscópicas e histopatológicas uterinas en vacas sometidas a aspiraciones foliculares

Transvaginal follicular aspiration guided by ultrasonography that helps to take advantage of the genetic potential of females considered superior. However, follicular aspiration is not an invasive technique for the reproductive tract in which it is used once the organ is punctured by a needle for several times. Therefore, the present work aims to characterize macroscopic and histopathological changes in the uterus of bovine females submitted to a sequence of follicular punctures, created in the Uberaba-MG region. Histopathological examination revealed 56 lesions: 32% (18/56) acute endometritis, 7% (4/56) chronic endometritis, 29% (16/56) fibroma, 5% (3/56) panmetritis, 7% (4/56) cystic endometritis, 11% (6/56) leiomyoma, 2% (1/56) mucopurulent endometritis, 4% (2/56) muscular hyaline degeneration of the artery wall, 2% (1/56) polyp Endometrium, 2% (1/56) suppurative endometritis. It is concluded that these pathologies caused by follicular aspiration continue to promote subfertility in these animals.

Effect of treatment of Bos indicus heifers with progesterone 0, 3 and 6 days after follicular aspiration on follicular dynamics and the timing of oestrus and ovulation

Synchronisation of wave emergence is used to synchronise oestrus in cattle. The aim of this study was to determine if treatment with high concentrations of progesterone in Bos indicus heifers for 3 days would synchronise new wave emergence when treatment commenced at early, mid and late stages of follicular development. Heifers were treated with a sc silicone implant containing norgestomet from Days -7 to 9 and cloprostenol (IM) on Days -7 and -2. All follicles > 4 mm in diameter were removed by transvaginal follicular aspiration either on Days 0 (Experiment 1), 3 (Experiment 2) or 6 (Experiment 3). From Days 6 to 9 every heifer was treated with two intravaginal progesterone releasing inserts that each contained either no progesterone (Control, n = 8/experiment) or 3.12 g of progesterone (n = 8/experiment). Ovarian follicular development was monitored at least once daily following aspiration until oestrus and ovulation. In each experiment, treatment with progesterone significantly increased concentrations of progesterone in plasma from Days 6 to 9 compared to Control heifers. It also significantly delayed the day of emergence of the ovulatory follicle (1.6 ± 0.6 vs 8.6 ± 0.3; 4.1 ± 0.1 vs 8.6 ± 0.2; 7.0 ± 0.0 vs 9.3 ± 0.4, for Control vs progesterone treated heifers, respectively in Experiments 1 to 3) and the interval from implant removal to oestrus and ovulation. In conclusion, treatment with high concentrations of progesterone can synchronise wave emergence in Bos indicus heifers when administered at early, mid and late stages of follicular development.

Use of different pressures for transvaginal follicular aspiration in mares

ABSTRACT The success of transvaginal follicular aspiration in mares can be influenced by several factors, such as vacuum pump pressure levels. The present study aimed to investigate the effect of different negative pressures (150, 280 and 400mmHg) of the vacuum pump on the oocyte recovery in the mares. The mares (n=10) were undergoing follicular aspiration using three different negative pressures for three consecutive estrous cycles as follows: G150 = 150mmHg (n = 10); G280 = 280mmHg (n = 10); G400 = 400mmHg (n = 10). Every estrous cycle, the group that the mare would participate was drawn, and each animal participated once in each group. Only preovulatory follicle was used, about 30 to 36 hours after application of hCG. To compare the results, the chi-square test was used (5% significance) and Fisher exact test, when recommended. Thirty preovulatory follicles (diameter 36.1±1.80mm) were aspirated and ten oocytes were recovered (33.3%). There was no statistical difference between the experimental groups (p=0.59). Thus, accord to the results observed in this study, we could conclude that the negative pressure of the vacuum pump used was not efficient to increase oocyte recovery.

Impact of acute metabolic acidosis on the acid-base balance in follicular fluid and blood in dairy cattle

Acid-base balance is one of the most vigorously regulated variables of the body, including genital organs. Subacute ruminal acidosis is a common disturbance in dairy cows that disturbs several biochemical indices in the blood, cerebrospinal fluid, and urine. The possible negative effects of metabolic acidosis on the follicular fluid (FF) composition and, subsequently, on oocyte quality, are not fully elucidated. This study aimed to evaluate the changes in acid-base balance (ABB) in FF and blood during acute metabolic acidosis in dairy heifers. Ten Holstein heifers were stimulated with FSH in eight decreasing doses at 12-hour intervals (D0–D3). Acidosis was induced by oral administration of sucrose at 9 g/kg of body weight, dissolved in 10 L of warm tap water, at D3. Samples were collected from each cow at 0, 8, 12, 16, 24, 32, 40, and 48 hours after treatment. Samples of FF, obtained by transvaginal follicular aspiration, and peripheral blood were examined for ABB parameters: pH, pCO2, pO2, HCO3−, and base excess (BE). A significant decrease in pH, HCO3−, and BE values in the blood, as well as FF, occurred after sucrose treatment. The lowest pH values occurred in blood at 16 hours, and in FF at 24 hours, after treatment (7.30 ± 0.05 and 7.33 ± 0.05, respectively). The lowest HCO3− values in blood (18.75 ± 3.2 mmol/L) and FF (18.07 ± 2.84 mmol/L) occurred 24 hours after treatment, as did the lowest BE values (−6.61 ± 3.7 mmol/L and −7.53 ± 3.89 mmol/L, in blood and FF, respectively). Significant correlations for HCO3− (r = 0.928), BE (r = 0.946), pH (r = 0.889), and pCO2 (r = 0.522) existed between blood and FF samples. The results demonstrated that metabolic acute acidosis substantially influences the characteristics of both serum and FF.

Comparison of PUFA Profiles in the Blood and in Follicular Fluid and its Association with Follicular Dynamics after PGF2α Induced Luteolysis in Dairy Cows

Abstract The objectives of the present study were to examine the fatty acid (FA) profiles in serum and in the follicular fluid (FF) and the association between polyunsaturated fatty acid level (PUFA) and follicular growth dynamics following induced luteolysis in dairy cows. A total of 29 dairy cows (CL&gt;25mm, follicle≈15mm) at d0 (start of the experiment) were submitted to ultrasound guided transvaginal follicular aspiration for FF collection from the largest follicle and were injected with 500 μg of cloprostenol. The cows were subdivided into Group A1 (n=11) and Group A2 (n=8) resuming follicular growth either from a secondary follicle less than or larger than 8.5mm, respectively, present at the moment of aspiration and Group A0 (n=10) not resuming follicular growth. Follicular development was monitored daily by ultrasonography until the next dominant follicle reached ≈15mm and was subsequently punctured in Group A1 and A2 (d1). Serum and FF samples for FA determination were taken at d0 from all cows and at d1 in Group A1 and A2. No differences were observed between the FA profile in serum nor in FF between sampling days. Regarding the PUFA levels, the serum linoleic acid (C18:2n6) levels at d0 and d1 were significantly higher than in FF, while alpha linolenic acid (C18:3n3) was lower in the serum than in FF, both at d0 and d1. At d0, a tendency for negative correlation between serum and the FF C18:2n6 with subsequent daily follicular growth rate was observed, while, at d1 there was a strong negative correlation between the serum C18:2n6 and daily growth rate (r=−0.71; p=0.0006). The present study revealed similarities of the FA profiles in the serum and in the FF and association between serum and FF PUFA content with the follicular dynamics after induced luteolysis.

Dynamics of in vitro maturation of equine oocytes recovered by transvaginal follicular aspiration

Dynamics of in vitro maturation of equine oocytes recovered by transvaginal follicular aspiration

Clinical and pathological presentation of squamous metaplasia of the rete ovarii in a Zebu cow

Squamous metaplasia of rete ovarii is characterized by replacement of the normal cuboidal epithelium of rete ovarii by a keratinized stratified scamous epithelium, leading to accumulation of keratinized material within the tubules and cystic dilatation of rete ovarii. The present study decribes a case of scamous metaplasia of rete ovarii in a 10 year old Zebu cow, including clinical, surgical, ultrasonographic, histopathological and hormonal findings. At first ultrasound examination the cow had lightly asymmetric ovaries with follicles presenting echogenicity similar to luteinized follicular cysts. After transvaginal follicular aspiration creamy yellowish sanguineous-purulent content was recovered. After unilateral ovariectomy the ovary was sectioned and brownish viscous material drained from cystic cavity. Histopathology confirmed the diagnosid of squamous metaplasia of the rete ovarii. Progesterone concentrations assessed by chemiluminescent enzyme immunoassay within different time periods after ovariectomy showed that pathology did not compromise normal luteal ovarian activity in a contralateral reminiscent ovary.

Effect of Follicular Flushing During Oocyte Retrievalon Clinical Outcome of Assisted Reproductive Technology

Objective:is to determine whether follicular aspiration and flushing increase the number of oocytes yield and pregnancy outcome over aspiration alone in women undergoing ICSI. Study design: prospective randomized controlled trial. One hundred eighty five infertile women who underwent ICSI were included in the study. They were randomized into two groups 92 cases in (aspiration and flushing group) and 93 cases in (aspiration only group),during the period from September 2011 to September 2013. Intervention(S):Controlled ovarian hyperstimulation using long GnRH agonist was the standard protocol, hCGadministrated 10000 iu when three or more follicles were at least 18 mm in largest diameter, Trans-vaginal follicular aspiration performed 34-36 hours after hCG trigger.In the aspiration alone group, a 16 gauge single lumen needle used, with suction continue until a small amount of blood stained fluid appeared in the tubing or flow stop, When flushing accompany aspiration of follicular fluid in the study group, the same needle used with a double-way tap allowing flushing of (2 ml) of embty follicleby Earl's medium till oocyte retrieved or maximum two times. Results:The study observed 60.5 % oocyte recovery rate with aspiration only compared with 80.9 % with follicular aspiration and flushing.Operative time (minutes) was significantly longer among flushing group,the retrieval time was 1.3 fold higher among those undergoing follicular flushing. Pregnancy was non-significantly more frequent among flushing. Implantation rates non-significantly more frequent among flushing group than non-flushing group (31.6% versus 26.3%, P= 0.424) and ongoing clinical pregnancy non-significantly more frequent among flushing group (27.4% versus 21.1% , P= 0.31). Conclusion, flushing non-significantly increase implantation and clinical pregnancy outcome and associated with a significant increase in the procedure time for oocyte retrieval, so patient groups where a small number of oocytes are available for retrieval may represent patients most likely to benefit from follicle flushing as only one extra oocyte may affect the outcome.