Cumulus–oocyte Complexes Collection Research Articles

Enhancing the quality of inferior oocytes of buffalo for in vitro embryo production: The impact of melatonin on maturation, SCNT, and epigenetic modifications

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10-9M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10-9M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10-9M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10-9M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning.

Comparison of superstimulatory protocols with different doses of eCG on ovarian response and oocyte recovery by follicular aspiration in llamas (Lama glama).

The objective of this study was to evaluate the effect of three doses of equine chorionic gonadotropin (eCG) for ovarian superstimulation on ovarian response, follicular development and cumulus-oocyte complexes (COCs) collection in llamas. For this purpose, eighteen multiparous non-lactating adult (4-7 yo) female llamas with an average body condition of 2.8 (BCS 1-5) were submitted to a follicular ablation (FA) to induce a new follicular wave emergence. Two days after FA (Day 0), synchronized llamas were randomly allocated to three treatment groups (n = 6/group) and given 500, 750 and 1000 IU of eCG (Novormon®, Syntex, Buenos Aires, Argentina) per animal respectively to induce ovarian superstimulation. Transrectal ultrasonography were performed on Days 2, 4, and 6; and ovum pick up (OPU) was performed on Day 6. Data was evaluated by one-way analysis of variance (ANOVA), repeated measures ANOVA, and 2-tailed Chi-square. The average size (mm) of follicles was greater (p≤ 0.05) in the 1000 IU group compared to the other groups. There was a greater (p≤ 0.05) number of follicles ≥ 7 mm in the 1000 IU group compared to the 500 IU group. Number of COCs collected on Day 6 and the COC recovery rate were not different among groups. In conclusion, a single dose of 1000 IU of eCG induced the best ovarian response resulting in larger and greater number of follicles at the time of OPU.

Progesterone Promotes In Vitro Maturation of Domestic Dog Oocytes Leading to Successful Live Births.

Gene-edited dogs are promising models for biomedical research because they have hundreds of genetic diseases that are similar to humans. A common method for producing gene-edited dogs is assisted reproductive technology (ART) using in vivo oocytes or embryos, but it is much more inefficient and has a higher cost. ART for dogs has lagged mostly because of the lack of an efficient in vitro maturation system. Because early maturation of canine oocytes occurs in follicles with extremely high concentrations of progesterone (P4), we hypothesize that P4 has an important role during maturation. In this study, we obtained ovaries of female dogs and collected cumulus-oocyte complexes, which were cultured in vitro in microdrops containing different P4 concentrations (0, 10, 40, 100 or 200 µg/mL). We found that 40 µg/mL P4 produced the highest oocyte maturation rate (29.7% ± 7.1%, p &lt; 0.05). We also evaluated the quality of in vitro matured oocytes by in vitro fertilization and single-cell RNA sequencing, and both indicated an improvement in oocyte developmental potential. In conclusion, we successfully obtained the first live dogs using in vitro matured oocytes by adding P4 to optimize the in vitro maturation system of canine oocytes, and established a new and low-cost method to produce dogs via in vitro maturation and in vitro fertilization.

P-401 The impact of the recipients’ age in vitrified/warmed oocyte donation (OD) cycles

Abstract Study question Does female age affect reproductive outcomes in a vitrified/warmed OD program with fresh embryo transfer? Summary answer Oocyte donation performed below the age of 40 was associated with less favorable reproductive outcomes. What is known already It is well established that oocyte and embryo aneuploidy are mainly responsible for the age-related decline in female fertility. Previous studies evaluating the effect of maternal age on implantation potential in OD cycles generated controversial results. It remains unclear whether female age affects reproductive outcome independent of embryo quality. Study design, size, duration This is a retrospective, single tertiary center, cohort study analyzing a vitrified/warmed OD program over a ten-years period (February 2010-February 2020). More specifically, data from 491 unique oocyte recipients were included who performed a fresh embryo transfer after sharing (with at least two recipients) sibling oocytes from the same donor stimulation cycle. For all treatments ICSI was performed without preimplantation genetic testing. Participants/materials, setting, methods The association between recipient’s age and reproductive outcomes was investigated according to three age categories (&amp;lt;35/35-40/&amp;gt;40 years old). The primary outcome was live birth rate (LBR), while the secondary outcome was biochemical pregnancy rate (BPR). A multivariate regression model was developed adjusting for the following covariates (entered simultaneously): recipient’s BMI, recipient’s endometrial thickness, age of the oocyte donor, number of mature oocytes used for ICSI, embryo transfer stage and number of embryos transferred. Main results and the role of chance Mean age of donors was 28.6 years old(±4.1). Mean starting dose was 192.6 IU(±139.9) and duration of stimulation 11.0 days (±1.7). Mean number of collected cumulus oocyte complexes was 25.6(±9.6) and sibling oocytes were assigned to 2, 3 or more recipients in, respectively, 83.1%, 12.4% and 4.5% of cases. Mean age of recipients was 38.6 years old(±5.1), BMI 24.3 kg/m²(±4.1) and endometrial thickness 8.9mm(±2.1). ICSI was performed on a mean number of 6.5 (±2.0) warmed, intact, mature oocytes with a fertilization rate of 68.2% (±22.5). Cleavage-stage embryos were transferred in 91.0% of the recipients, with a double embryo transfer in 42.6%. 81.3% of embryos were scored as top-quality and a mean of 1.4 (±1.4) surplus embryos were available for additional vitrification. Overall BPR was 44.2%, clinical pregnancy rate 39.3% and LBR 31.0%. Categorizing according to recipient’s age showed a LBR of 24.3%,26.7% and 37.0% for, respectively, the group &amp;lt;35, 35-40 and &amp;gt;40 years old (p = 0.02). The multivariate GEE regression model confirmed the crude data showing that the age was a significant positive predictor of LBR [coefficient (-), 0.01,0.11, for group&amp;lt;35,35-40 and &amp;gt;40 years old, p = 0.05] and BPR [coefficient (-),0.008,0.15, for group &amp;lt;35,35-40 and &amp;gt;40 years old, p = 0.006]. Limitations, reasons for caution Sibling donor oocytes were used, but the retrospective nature of the study remains a reason for caution. Also, the fact that the majority of embryos were transferred at the cleavage stage is a limitation. Wider implications of the findings The observation that recipients below the age of 40 have lower reproductive success rates following oocyte donation deserves further attention. The need for oocyte donation at younger age might be associated with a less performant uterine/endometrial function due to underlying conditions of genetic and/or endocrine nature. Trial registration number B1432020000146

Strategies for oocyte collection and in vitro embryo production in free-roaming bison herds.

The study was conducted to test the feasibility of protocols for field collection of cumulus–oocyte complexes (COC) for in vitro embryo production (IVP) in wild bison. The study was done with captive wood bison during the anovulatory season. In Experiment 1, the efficiency of transvaginal ultrasound-guided COC collection was compared between bison restrained in a squeeze chute without sedation vs in lateral recumbency after chemical immobilization using a dart gun (n = 8/group). In Experiment 2, a 2 × 2 design was used to examine the effects of superstimulation treatment [single dose of equine chorionic gonodotrophin (eCG) vs multiple doses of follicle stimulating hormone (FSH)] and method of drug administration (manual injection vs field darting) on COC collection and IVP. In Experiment 1, no difference was detected between chute-restrained vs chemically immobilized groups in the time required to complete COC collections, the number of follicles aspirated (11.5 ± 1.9 vs 9.3 ± 1.8; P = 0.4) or the COC recovery rate [COC recovered/follicle aspirated; 58/92 (63%) vs 44/69 (64%); P = 0.9]. In Experiment 2, no differences were detected between superstimulation treatments (eCG vs FSH). The total number of follicles available for aspiration did not differ between manual injection and field darting (23.9 ± 2.7 vs 21.6 ± 1.9; P = 0.4). Compared with the random start unstimulated group, the embryo production rate was higher [18/132 (14%) vs 53/189 (28%); P = 0.04] after wave synchronization and superstimulation. Results suggest that COC collection is equally feasible in a recumbent position after chemical immobilization as those bison restrained in a standing position in a hydraulic chute. Ovarian superstimulation with a single-dose eCG protocol is as effective as a multiple-dose FSH protocol, and field darting is as effective as chute-side administration of superstimulation treatments. The strategies in the present study are ready to be incorporated into field collections in free-roaming bison herds.

Antral follicle counts and association with ovarian superstimulatory response to gonadotropins in prepubertal calves

Antral follicle count (AFC) repeatability at the time of follicular wave emergence and duration of gonadotropin treatment in calves with small and large AFC affects the superstimulatory response of follicles. In Study I, the individual AFC was determined, calves were ranked as having a small, medium or large AFC, and a second count was performed prior to FSH treatments. There was a positive association between the number of follicles ≥1 mm after the first and second counts (r = 0.4; P = 0.003). In Study II, calves with small and large AFC were administered pFSH for 4 or 7 days, pLH 20 h after last pFSH administration and cumulus-oocyte-complexes (COC) were collected. In calves having large as compared with small AFC, number of follicles ≥6 mm were greater (P = 0.01) and COC collected were greater (P = 0.001). The proportion of large-sized follicles (>9 mm) was greater in the 7-day than in the 4-day gonadotropin treatment group (56.4 ± 8.3 and27.8 ± 7.5 %, respectively; P = 0.01). In Study III, there was a positive association between AFC and number of follicles ≥6 mm at the time of COC collection (r = 0.6; P = 0.003). In summary, the number of follicles at the time of follicular wave emergence was associated with the number of follicles recruited during subsequent waves of follicular development and ovarian response following gonadotropin superstimulation. Calves with a large AFC had more COC collected than calves with a small AFC.

38 Developmental competence of bovine cumulus–oocyte complexes collected from cows fed rumen-protected methionine and lysine

Supplementation of rumen-protected amino acids (RPAA) has proven to be an effective tool to supply limiting AA in dairy diets. Methionine and lysine are the two most limiting AA for lactating dairy cows. Recently, it has been shown that methionine supplementation seems to affect pre-implantation embryos collected from superovulated cows enhancing their developmental competence because there is strong evidence that endogenous lipid reserves serve as an energy substrate (Acosta et al. 2016 Theriogenology 85, 1669–1679). Moreover, higher concentrations of methionine were determined in the follicular fluid of the first dominant follicle postpartum in cows supplemented with rumen-protected methionine and rumen-protected choline from 21 days before calving to 30 days postpartum and it was assumed that higher methionine concentrations in the follicular fluid could affect oocyte quality (Acosta et al. 2017 Theriogenology 96, 1–9). There is no information available so far regarding the effect of a combined methionine and lysine supplementation (each rumen-protected) on oocyte quality. Therefore, the objective of this study was to evaluate the effect of a combined methionine and lysine supplementation during early to mid-lactation on the developmental competence of oocytes collected from lactating dairy cows (days 0 to 100 p.p.). Thirty pregnant multiparous German Holstein dairy cows were grouped 3 weeks before their expected calving date, receiving identical diets. After calving, they were randomly allocated to 2 groups fed a total mixed ration supplemented with (N=14 cows; RPAA) or without (N=16 cows; CON) LysiGEMTM (encapsulated lysine; Kemin Industries) and Metasmart DryTM (isopropyl ester of the hydroxylated analogue of methionine adsorbed onto a silicon dioxide carrier; Adisseo). Starting from 45 days p.p., animals from both groups were submitted to an ovum pickup (OPU) session once a week for at least 8 weeks. Collected cumulus–oocyte complexes (COC) were subjected to a standard invitro production (IVP) protocol (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–10) including IVM, IVF, and invitro culture (IVC). Cleavage and developmental rates up to the morula/blastocyst stage were recorded on Days 3, 7, and 8. In total, 1211 follicles have been aspirated from RPAA animals compared with 1413 from CON animals, from which 742 and 885 COC were collected, respectively. The calculated recovery rate based on the number of aspirated follicles and collected COC was similar for both groups (61.3±29.4% vs. 62.6±33.5%). Cleavage and developmental rates based on 240 (RPAA group) and 299 (CON group) COC also showed similar results [RPAA: 84.1±5.9% (202/240), 18.3±4.4% (44/240), 18.8±4.7% (45/240); CON: 81.9±8.6% (245/299), 15.4±8.9% (46/299), 16.7±8.4% (50/299)]. In conclusion, supplementation of RPAA (methionine and lysine) had no beneficial effect on the developmental competence of COC obtained from these animals compared with those collected from cows fed the diet without RPAA supplementation.

22 Strategies for oocyte collection procedures in free-roaming bison herds

The study was conducted to test the feasibility of protocols for field collection of cumulus–oocyte complexes (COC) for invitro embryo production (IVP) in wild bison. The study was done with captive wood bison during the late anovulatory season (July). In Experiment 1, bison were assigned randomly to 2 groups (n=8/group) in which transvaginal ultrasound-guided COC collection was done in a chute without sedation or in lateral recumbency after chemical immobilization using a dart gun to compare collection efficiencies. In Experiment 2, a 2×2 design was used to examine the effects of superstimulation treatments [single-dose equine chorionic gonadotrophin (eCG) vs. multiple-dose FSH] and methods of drug administration (manual injection vs. field darting) on COC collection and IVP. Initial COC collection was done to serve as a non-superstimulated random start and to synchronize follicular wave emergence on the following day (Day 0; n=16). Half of the bison were given a single dose of 5000IU of eCG intramuscularly (IM) on Day −1. The other half were given 200mg of FSH IM on Day 0 and Day 2, followed by 2000IU human chorionic gonadotrophin IM on Day 4. Superstimulatory treatments were given by manual injection (chute restrained) to half of the bison in each group and by field darting in the other half. The COC were collected on Day 4 in the eCG group and Day 5 in the FSH group. Recovered COC were matured invitro for 25 to 28h at 38.8°C, fertilized (2×106 sperm mL−1) and co-incubated at 38.8°C in 5% O2, 5% CO2, and 90% N2 for 18h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O2, 5% CO2, and 90% N2. A commercially available IVF media set was utilised (IVF Bioscience). Data were compared by ANOVA and GLIMMIX. In Experiment 1, no difference was detected between chute-restrained vs. chemically immobilized groups in the time required to complete COC collection (6.9±1.0 vs. 8.9±1.0 min; P=0.2), the number of follicles aspirated (11.5±1.9 vs. 9.3±1.8; P=0.4), or the COC recovery rate [COC recovered/follicle aspirated; 58/92 (63%) vs. 44/69 (64%); P=0.9]. In Experiment 2, no differences were detected between superstimulation treatments (eCG vs. FSH) or method of drug administration (manual injection vs. field darting) for any endpoint. The number of medium (4.5–7.5 mm; 9.5±1.0 vs. 2.5±0.6; P&amp;lt;0.0001) and large (&amp;gt;8 mm; 8.6±0.8 vs. 3.4±1.5; P=0.004) follicles available was greater after superstimulation (groups combined) than without superstimulation (random start). Although there was no difference in the number of recovered compact COC between the superstimulated and non-superstimulated bison (8.9±1.3 vs. 6.2±1.7; P=0.2), the embryo production rate (number of embryos produced/number of COC matured) was greater after superstimulation than without superstimulation [54/189 (39%) vs. 19/108 (18%); P&amp;lt;0.05]. We conclude that COC collection in a field setting is feasible and minimum-handling superstimulation with a single dose of eCG is as effective as a multiple-dose FSH protocol for the invitro production of embryos in bison. This research was supported by NSERC and Vetoquinol.

The antioxidant dimethylthiourea improves IVF efficiency and decreases cumulus cell apoptosis in pigs.

Abattoir ovaries, which are the main source of oocytes for reproductive biotechnologies, arrive at the laboratory under ischaemic conditions. Reoxygenation generates reactive oxygen species (ROS) in ischaemic tissues, which could affect oocyte quality. The aim of this study was to evaluate the effect of supplementation of media with dimethylthiourea (DMTU) during the collection and washing of cumulus-oocyte complexes (COC) on ROS levels, COC apoptosis and oocyte nuclear and cytoplasmic maturation. Thus, the collection (TCM-199) and washing (TCM-199 with 10% porcine follicular fluid, sodium pyruvate and antibiotics) media were supplemented with 1 and 10mM DMTU. In the control group, the media were not supplemented with DMTU. Intracellular ROS levels decreased significantly in the DMTU-treated groups (P<0.05). Although no effects on rate of nuclear maturation were observed, DMTU significantly increased sperm penetration rates without increasing polyspermy (P<0.05). The addition of 10mM DMTU to the collection and washing media enhanced IVF efficiency. DMTU did not modify the early or late apoptosis of oocytes. Both concentrations of DMTU significantly increased viability and decreased the apoptosis of cumulus cells (P<0.05). These results suggest that the addition of 1 or 10mM of DMTU to the media during the collection and washing of porcine COCs is useful for decreasing cumulus apoptosis mediated by ROS and for optimising the IVF of porcine oocytes.

135 Effect of Different Bovine Breeds on Efficiency of Embryo Production by Ovum Pick-Up-IVF

Production of bovine embryos by ovum pick-up (OPU)-IVF has increased in recent years. However, numerous factors affect efficiencies of embryo production using this technology. This study was investigated the effects of different breeds on embryo production by OPU-IVF. In total, 98 OPU-IVF sessions were conducted on 41 Holstein and 27 Japanese Black cows from February 2015 to May 2017. The collected cumulus–oocyte complexes (COC) were matured for 22 h. After co-culture of COC with sperm for 6 h, the presumptive zygotes were washed and denuded by pipetting. Those zygotes were then cultured in CR1aa medium supplemented with 5% calf serum for 9 days in a micro-well culture dish. Blastocyst formation rates were analysed at 9 days post-insemination (hpi). The kinetics of embryo development was observed at 27, 31, and 55 hpi. Four factors were considered for selecting embryos to predict pregnancy competence: (1) timing of first cleavage; (2) formation of 2 blastomeres after first cleavage at 31 hpi; (3) absence of fragments after first cleavage at 31 hpi; (4) formation of 8 or more blastomeres at 55 hpi. The quality of blastocysts was compared with the proportions of embryos that were selected based on these 4 factors. Data were analysed by the Chi-squared test with Yates’ correction and Student’s t-test. Total numbers of COC from Holstein or Japanese Black cows were 1330 (n = 51) and 1543 (n = 47), respectively. However, no differences were observed in the numbers of collected and cultured COC per OPU session between Holstein (26.1 ± 20.1 and 21.7 ± 20.1) and Japanese Black (32.8 ± 24.7 and 28.7 ± 22.7) cows. The percentage of COC linked into Grade 1, 2, or 3 was 47.6, 32.1, and 10.7% for Japanese Black and 45.8, 25.6, and 12.2% for Holstein cows, respectively. The proportion of COC with expanded cumulus cells was significantly higher (P &lt; 0.01) in Holstein (12.7%) than in Japanese Black (7.7%) cows. The number of blastocyst and transferable embryo production per OPU session in Holstein (7.5 ± 8.3 and 7.3 ± 7.7) cows was not different from that in Japanese Black (10.6 ± 9.3 and 9.8 ± 9.0) cows. Moreover, there was no difference in blastocyst formation rates between Japanese Black (36.8%) and Holstein (34.8%) cows. Percentages of embryos selected by factors 1 to 4 were 42.6, 66.8, 59.1, and 54.4% in Holstein, and 54.6, 83.2, 72.2, and 75.2% in Japanese Black cows, respectively. Proportions of blastocysts in Holstein and Japanese Black cows selected by a combination of all 4 factors were 21.6 and 36.1%, respectively. There were significant differences (P &lt; 0.01) between the 2 breeds in terms of proportion of blastocysts selected based on individual factors as well as a combination of all 4 factors. These results suggested that the efficiency of embryo production did not differ between Japanese Black and Holstein cows. However, the quality of embryos selected by a combination of 4 factors was significantly higher in Japanese Black than in Holstein cows.

80 Cumulus Cells Reflect the Success of IVF of Porcine Oocytes Treated with Dimethylthiourea

The aim of this study was to evaluate the effect of the antioxidant dimethylthiourea (DMTU) as a supplement during the collection and washing of cumulus–oocyte complexes (COC) on the apoptosis in cumulus cells (CC) and the competition for oocyte development in vitro. The COC were obtained by follicular aspiration. The aspiration and washing medium was TCM-199 with 10% porcine follicular fluid, 0.3 mM sodium pyruvate, and penicillin/streptomycin. Treated groups were supplemented with 2 or 20 mM DMTU. After 44 h of in vitro maturation (IVM), the percentage of nuclear maturation was determined by staining of the denuded oocytes with Hoechst 33442 (n = 357). After 18 h of in vitro fertilization (IVF) with 15°C refrigerated spermatozoa from boars of proven fertility, the suspected zygotes were stained with Hoechst 33342 to evaluate the percentages of sperm penetration (SP), monospermic penetration (MP), male pronucleus formation (MPN), and IVF efficiency (monospermic oocytes/total oocytes) (n = 260). In post-IVM CC, flow cytometry was performed after staining with annexin V-propidium iodide to evaluate apoptosis and viability (n = 90,000). Data were analysed with Flowing V 2.5.1 (http://flowingsoftware.btk.fi/). A comparison of proportion among categories was used (Fisher’s exact test), and significant differences were determined at P &lt; 0.05. No effects were observed in the percentages of nuclear maturation. Treatments with DMTU significantly increased the SP (Control: 35%; 2 mM: 72.9%; 20 mM: 67.8%) without modifying the MP. Although in both treatments, the amount of MPN was always greater than in the control, the percentages of MPN formation did not show significant differences. The 20 mM treatment significantly increased the efficiency of IVF (control: 22.2%; 2 mM: 31.9%; 20 mM: 45.7%). In the CC of the treated groups, a significant increase in viability (control: 7.4%; 2 mM: 12.2%; 20 mM: 14.7%) and a decrease in early apoptosis (control: 60.4%; 2 mM: 55.5%; 20 mM: 57.1%) were observed. Late apoptosis decreased in the 20 mM treatment (27.3%) compared with the other experimental groups (control: 31.3%; 2 mM: 31.2%). The addition of DMTU during COC collection and washing reduce the ROS-mediated apoptosis in CC and optimize the efficiency of IVF. Besides, CC quality reflects the effect of DMTU on oocytes, so viability and apoptosis analysis on CC constitutes an interesting noninvasive method to assess oocyte competence.

Effect of ovarian types and collection techniques on the number of follicles and the quality of cumulus-oocyte-complexes in cow

This experiment was conducted for evaluation of bovine slaughterhouse ovary, follicles, and cumulus-oocyte-complexes (COCs) and to compare the effect of collection techniques on the recovery rate of COCs. The collected slaughterhouse ovaries was classified as corpus luteum present (CL+) and corpus luteum absent (CL-) groups. It was found that 62.5% of the ovaries collected were CL- type and 37.5% were CL+ type. For collection of COCs, blunt dissection and aspiration techniques were performed and number of follicles collected was recorded. The higher number of follicles were dissected and aspirated from CL - ovaries (11.2±1.8and 37.8±14.9, respectively) compared to CL+ ovaries 10.5±1.5and 28.3±15.6, respectively). The follicular materials collected from both techniques were observed under microscope to categorize the COCs as A (oocyte surrounded with cumulous cells homogenously), B (oocyte surrounded with cumulous cells partially), C (oocyte not surrounded at all by cumulous cells) and D (degeneration observed both in oocyte and cumulous cells). Grade A and grade B were classified as normal and grade C and grade D were considered as abnormal COCs. The result indicated that ovaries having no CL contributing more total number of COCs per ovary (6.8±1.0) and also contributing higher normal COCs (5.7±0.9) than that of ovaries with CL (6.0±2.0 and 4.5±1.5, respectively) in blunt dissection technique. But same trend of result was not found in aspiration technique. Similarly, higher percentage of COCs recovery rate was also recorded in blunt dissection (61.6±4.6% vs 16.5±4.9%, on total basis) than aspiration (48.6±2.9% vs 11.7±4.1%, on normal basis) technique. So, ovaries without CL and blunt dissection technique found more suitable for harvesting the higher number and superior quality of COCs for extending the in vitro embryo production experiment.Bang. J. Anim. Sci. 2016. 45 (3): 10-16

136 IMPACT OF SELECTION SYSTEM BY KINETICS ON THE EARLY EMBRYONIC DEVELOPMENT IN BOVINE OVUM PICKUP-IVF EMBRYOS

Recently, in vitro-produced (IVP) embryos have been increasingly produced using ovum pickup (OPU) and IVF in cows worldwide. However, the conception rate of IVP embryos is lower than that of in vivo-derived embryos. This study was conducted to determine the proportion of embryos that led to a high conception rate when the embryos were selected according to the 4 predicting factors. A total of 30 Holstein and 20 Japanese Black cows were used, and 81 OPU-IVF sessions were performed from October 2014 to May 2016. The collected cumulus-oocyte complexes (COC) were cultured for 22 h. Capacitated sperm (at a final concentration of 5 × 106 spermatozoa/mL) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% calf serum by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). The kinetics of embryo development was observed at 27, 31, and 55 h post-insemination (hpi). The 4 factors used to select embryos were as follows: (1) time at which first cleavage occurred (less than 27 hpi, or less than 31 hpi, in case any of the zygotes did not cleave at 27 hpi in each culture dish); (2) 2 blastomeres after first cleavage at 31 hpi; (3) absence of fragments after first cleavage at 31 hpi; and (4) 8 or more blastomeres at 55 hpi. The number of blastocysts was analysed at 7, 8, and 9 days post IVF. Additionally, the number of produced embryos that could be used for embryo transfer (ET) was determined. The data were analysed using the chi-square test. The total numbers of blastocysts and produced embryos were 615 and 503, respectively. The numbers of blastocysts and produced embryos selected using the combination of factors 1 to 4 were 200 (32.5%) and 169 (27.5%), respectively. The numbers of blastocysts and produced embryos selected using factor 1 were 397 (64.6%) and 340 (67.6%), using factor 2 were 445 (71.3%) and 378 (75.1%), using factor 3 were 364 (81.8%) and 307 (81.2%), and using factor 4 were 374 (60.8%) and 308 (61.2%), respectively. The numbers of blastocysts and produced embryos that were rejected using a combination of factors 1 to 4 were 123 (27.5%) and 90 (17.9%), respectively. The conception rate of fresh embryo transfer was 46.6% (n = 73). We found that the conception rate of the embryos selected using factors 1 to 4 was significantly (P &lt; 0.05) higher than that of embryos without one factor or more [60.0% (n = 35) v. 29.4% (n = 34)]. These results show the applicability and efficiency of the 4 factors for producing embryos with a high competence for conception.

Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation.

Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 μg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse oocytes. This may occur by its beneficial effect in increasing GSH concentrations in MII oocytes.

A systemic review on ultrasonographic applications in camels

Ultrasound is widely accepted as a safe noninvasive diagnostic imaging technique in animals and human. This review aims to shed light on the current applications and future prospect of ultrasonography in camels. To date, ultrasonography has been used efficiently to study the ovarian status in she camels such as; follicular wave, spontaneous ovulation, optimum time for mating, ovarian vasculature, superovulatory response, ovarian follicular dynamics, ovarian follicular wave synchronisation and follicular deviation. Moreover, it has been applied for collection of cumulus oocyte complexes, pregnancy diagnosis, foetometry, foetal sexing, embryo transfer programmes, assessment of somatic cell nuclear transfer and evaluation of the quality and developmental ability of dromedary embryos. Uterine involution and various reproductive disorders such as; early embryonic death, endometritis, vaginal adhesions, ovarian cysts and ovarian hydrobursitis have been diagnosed by ultrasound. In male camels, ultrasonography is a useful tool in studying the developmental changes of testes and pelvic genitalia including; bulbourethral gland, prostate, and pelvic urethra and predicting puberty and future fertility. Normal pleura, heart, fore stomach, liver, small and large intestines, kidney, eye, udder and teat, foot, carpal and tarsal joints have been successfully imaged. However, very limited affections of these structures including; infectious pleuropneumonia, peritonitis, trypanosomiasis, John's disease, intestinal obstruction and ruptured urinary bladder have been diagnosed ultrasonographically in camels. Therefore, ultrasonographic application in camels, compared to other farm animals, is still limited. In conclusion, ultrasonography is untapped in camel practice however, it can offer veterinarians the opportunity for more precise diagnosis and treatment of numerous disorders.

164 EFFECTS OF KINETICS AND MORPHOLOGY ON EARLY EMBRYONIC DEVELOPMENT IN BOVINE OPU-IVF EMBRYOS

In recent years, the use of ovum pick up (OPU) and IVF for embryo production has increased worldwide; however, the conception rate of embryo transfer is lower for OPU-IVF embryos than for in vivo-derived embryos. This study aimed to determine the efficacy of embryo selection by a 3-step observation method by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). In this study, 9 Holstein and 15 Japanese Black cows were used, and the OPU-IVF was conducted from October 2014 to May 2015. The collected cumulus-oocyte complexes (COC) were cultured for 22 h in 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH. Sperm (at a final concentration of 5 × 106 spermatozoa mL–1) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% CS by using a micro-well culture dish. Kinetics and morphology were observed at 27, 31, and 55 h post-insemination (hpi). The presumptive zygotes were divided to 3 groups (more than 2 cells, 2 cells, and no cleavage) at 27 and 31 hpi. Then, embryos at the 2-cell stage at 31 hpi were divided into 2 groups: 2-cell with normal cleavage and 2-cell embryos with abnormal cleavage (unequal cleavage, 2-cell with fragments, and 2-cell with protrusion). Subsequently, embryos were classified as 8-cell and more than 8 cell, or less than 8 cell at 55 hpi. The blastocyst rate (BL%) was analysed at 7, 8, and 9 days post IVF. Embryos selected by the 3-step observation method were used for fresh embryo transfer. The data were analysed by chi-squared test. In total, 856 oocytes were collected by OPU and 633 oocytes were cultured, of which 39.7% (263/663) developed to the blastocyst stage. The BL% of 2-cell embryos (72.5%, 116/160) was significantly higher (P &lt; 0.01) than that of no cleavage (47.0%, 117/249) at 27 hpi. The BL% of 2-cell (65.4%, 206/315) and more than 2-cell (53.0%, 35/66) was significantly higher (P &lt; 0.01 and P &lt; 0.05) than that of embryos divided as no cleavage (25.9%, 22/85) at 31 hpi. The BL% was not significantly different between 2-cell with normal cleavage (68.5%, 172/251) and abnormal cleavage (53.1%, 34/64). The BL% of 8-cell and more than 8-cell stage (72.8%, 182/250) was significantly higher (P &lt; 0.01) than that of embryos with less than 8 cells (38.8%, 81/209) at 55 hpi. Overall, 2-cell embryos at 27 hpi, 2-cell embryos with normal cleavage at 31 hpi, and 8-cell and more than 8 cell at 55 hpi showed the highest BL% (82.1%, 78/91). The conception rate was higher for following the selected fresh embryo transfer that was 70.6% (12/17) than average of in vitro fertilization embryos transfer that was 40.0%. These results demonstrate that the 3-step observation method used in this study can be effectively applied for the selection of IVF embryos that have a strong ability to develop into blastocysts and high competence for conception.

363 EXTENDING THE IN VIVO MATURATION TIME TO PERMIT FLEXIBLE TIMING OF OOCYTE COLLECTION IN SUPERSTIMULATED WOOD BISON (BISON BISON ATHABASCAE)

Wood bison are a species threatened by endemic brucellosis and tuberculosis. Reproductive technologies are being developed in an effort to ensure the genetic diversity of wild wood bison, and to prevent disease transmission to healthy bison, livestock, and humans. For the purposes of IVF, recent results revealed that cumulus cell expansion was more extensive in in vivo- v. in vitro-matured cumulus-oocyte complexes (COC), and more oocytes reached maturity after 30 v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2013 Reprod. Fert. Develop. 25, 283). An experiment was designed to determine the effects of an additional 4 h of in vivo maturation on follicle development, unwanted ovulation, and COC collection efficiency. Wood bison cows (n = 28) underwent transvaginal ultrasound-guided follicle ablation to induce emergence of a new follicular wave (Day 0 = day of wave emergence, 1 day after ablation) during the non-breeding season. Bison were given FSH diluted in hyaluronan IM on Days 0 (300 mg) and 2 (100 mg), and 2500 IU hCG IM on Day 4. Bison were then assigned randomly to 2 groups (n = 14 per group) in which transvaginal oocyte collection was done at either 30 or 34 h after hCG treatment. The number and size of follicles available for aspiration (i.e. = 5 mm) was compared between groups by Student's t-test. Binomial data (COC collection rate and ovulation rate) were compared by chi-square, and the proportion of cows that ovulated was compared using a Fisher's exact test. Ovulation was defined as the sudden disappearance of follicles ≥10 mm from the hCG treatment to the time of COC collection. The numbers of follicles ≥5 mm and ≥10 mm at the time of COC collection were not different between the 30 and 34 h groups (19.0 ± 1.4 v. 17.4 ± 2.4, and 9.5 ± 1.2 v. 7.7 ± 1.8), nor was the average size of follicles = 5 mm (9.9 ± 0.2 v. 9.8 ± 0.2 mm). The number of follicles aspirated was similar between the 30 and 34 h groups (16.4 ± 1.4 v. 13.4 ± 2.1), but the pre-collection ovulation rate was lower in the 30 h group (12/89 [13.5%] v. 47/147, [32.0%]; P = 0.003), as was the proportion of bison that ovulated (3/14 v. 10/14, P = 0.02). The COC collection rate was lower in the 30 v. 34 h group (64.3% v. 78.2%; P = 0.003), but the total number of COC collected per bison was similar (10.6 ± 1.7 v. 10.5 ± 1.5). Although waiting for 34 h before COC collection resulted in a larger proportion of unwanted ovulations, a greater collection efficiency in the 34 h group resulted in a similar number of COC collected per bison. We conclude that the 30 to 34 h in vivo maturation window provides flexibility for the purposes of oocyte collection and immediate in vitro fertilization in wood bison.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).

174 RECOVERY OF LONG-BEAKED COMMON DOLPHIN OOCYTES FROM KOREAN WATERS

The aims of this study were to collect oocytes from ovaries of Delphinus capensis (long-beaked common dolphin) and to examine the morphological characteristics of their reproductive organ. Female genital organs were collected from 2 incidentally caught Delphinus capensis (ID-96: estimated 5–6 years, and ID-97: estimated 7–8 years). Collected ovaries were transported to the laboratory over 3.5 h in 0.9% saline at 39°C. To perform assisted reproductive techniques in wild animals, it is important to maximize the number of oocyte recovered per ovary. In this study, Delphinus capensis oocytes were collected by 2 methods: (1) aspiration of visible follicles (2 to 3 mm in diameter) and (2) remnant dissection, in which the ovary is finely dissected. The collected cumulus–oocyte complexes (COC) were separated by ovary of origin and collection method. Oocytes were subjected to in vitro maturation (IVM). Immature oocytes were placed into IVM medium [TCM-199 supplemented with 10% (vol/vol) fetal bovine serum, 0.5 IU mL–1 LH, and 0.5 IU mL–1 porcine FSH] in the 4-well dish for 120 h at 37°C in 5% CO2 in a humidified atmosphere. The uterus type was observed to be a uterus bicornis. Morphology of ovary was similar to the bovine ovary among land animals. In total, 114 oocytes were obtained. From the ovary of ID-97, the number of oocytes recovered by aspiration and ovary dissection were 15 and 57, respectively. However, only 42 oocytes were recovered by ovary dissection from the ovary of ID-96. The number of oocytes recovered per ovary by dissection (49.5 ± 7.5) was higher than that by the aspiration (7.5 ± 7.5) method, but not significantly different (P = 0.06). For accurate statistical analysis, additional trials will be performed. Most oocytes had either no cumulus cells or had only 1 to 2 layers attached to the zona pellucida enclosing a homogeneous cytoplasm. After IVM, all oocytes were at the metaphase I stage, with even fewer attached cumulus cells than at recovery. To summarise, we describe the gross morphology of the female reproductive tract of the long-beaked common dolphin and determined that dissection of the ovary is required for maximal recovery of oocytes. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Superstimulatory response and oocyte collection in North American bison during the non-breeding season

A 2×2 design was used to compare the ovarian response and oocyte collection characteristics in bison given a superstimulatory dose of eCG or FSH, with or without a follow-up dose of LH. Follicular wave emergence was synchronized by follicle ablation (Day −1) and bison were assigned randomly to two superstimulatory treatment groups (n=10 per group): (i) a single intramuscular dose of 2500IU of eCG given on Day 0, or (ii) two subcutaneous doses of 200mg of FSH given on Days 0 and 2. On Day 4, 200mg of LH was given intramuscularly in 5 bison in each superstimulatory treatment group. The study was done in two replicates (n=20 per replicate) involving a crossover design so that each animal was given the opposite superstimulatory treatment (eCG or FSH) during successive replicates. Cumulus-oocyte complexes (COC) were collected by transvaginal ultrasound-guided follicle aspiration, and were classified according to morphologic attributes as compact, expanded, or denuded. Superstimulatory treatment with FSH (vs. eCG) resulted in the development of more follicles ≥5mm (14.2±1.41 vs. 8.2±0.67; P<0.05; mean±SEM), and more follicles aspirated (12.4±1.3 vs. 6.3±0.6; P<0.04). Follow-up treatment with LH (vs. no LH) resulted in a greater proportion of expanded COC (37% vs. 15%; P<0.05), and a tendency for a higher COC collection rate (61% vs. 54%; P=0.08). In summary, superstimulation with FSH (vs. eCG) resulted in twice as many follicles available for aspiration and nearly twice as many COC collected in bison during the anovulatory season, and follow-up treatment with LH increased the proportion of expanded COC collected.

252 EFFECT OF SUPERSTIMULATORY TREATMENT TO DONOR COWS IN OXYGEN CONSUMPTION OF MATURED OOCYTES

We previously reported that follicular wave synchronization and follicular growth treatment (FGT) before ovum pick-up (OPU) were effective in improving oocyte competence, which was associated with an increase in related embryos obtained by somatic cell nuclear transfer (Sugimura et al. 2012 Cell. Reprogram. 14, 29–37). However, oxygen consumption in oocytes remained unknown. The present study was designed to examine the differences in oxygen consumption between bovine oocytes obtained by OPU with or without FGT after in vitro maturation. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two OPU sessions were conducted in each cow to collect immature oocytes, as described by Sugimura et al. (2012). The first OPU session (OPU group) was performed in cows on arbitrary days of the oestrous cycle, using a 7.5-MHz linear transducer with the needle connected to an ultrasound scanner. Follicles larger than 8 mm in diameter were then aspirated and a controlled internal drug release device (CIDR) was inserted on Day 5 (the day of the first OPU session = Day 0). Then 30 Armour units (AU) of FSH (Antrin, Kyoritsu Seiyaku, Tokyo, Japan) was administrated to cows twice a day from Day 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 AU day–1). Cloprostenol (prostaglandin F2α; 0.75 mg) was administered in the morning of Day 9. The second OPU session (FGT-OPU group) was performed 48 h after prostaglandin F2α administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected cumulus–oocyte complexes in the OPU and FGT-OPU groups were matured in vitro as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. To collect in vivo-matured oocytes (control group), the CIDR was inserted into the cows on arbitrary days of the oestrous cycle (= Day 0), and oestradiol benzoate (0.8 mg) was administered on Day 1. The cows received the FGT treatment (as described above) from Day 6 to 10; however, the CIDR was removed in the evening of Day 8. Buserelin (gonadotropin-releasing hormone; 200 µg) was then administrated in the morning of Day 10, and OPU was performed at 24 h after gonadotropin-releasing hormone administration (Day 11). Oxygen consumption of matured oocytes was measured noninvasively with a scanning electron microscopy system (HV-405SP; Hokuto Denko Co., Tokyo, Japan). Data were analysed by ANOVA followed by a Tukey-Kramer test. There was no difference in the mean oxygen consumption between the FGT-OPU group (0.34 ± 0.02 × 10–14 mol–1, mean ± SEM) and control group (0.40 ± 0.01 × 10–14 mol–1). However, oxygen consumption in the FGT-OPU and control groups was significantly lower (P &lt; 0.01) than that in the OPU group (0.50 ± 0.02 × 10–14 mol–1). These results revealed significantly lower oxygen consumption in OPU-derived in vitro-matured bovine oocytes after FGT treatment compared with those obtained without FGT treatment. Oxygen consumption of oocytes obtained from FGT-OPU was similar to that of in vivo-matured oocytes, which may reflect their cytoplasmic maturation status with high developmental competence.