Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi

This study was aimed to determine the effect of urea in maturation medium on in vitro oocyte maturation rate. The medium used was TCM-199 added with Hepes, NaHCO3, Kanamycin 0.15 IU/mL, PMSG, 0.15 IU/mL hCG, and 10% FBS. Cumulus oocyte complexes (COCs) of cows derived from follicle aspiration were divided into three groups. In control group (P0), the COCs were matured in vitro in a maturation medium without urea addition, meanwhile in the P1 and P2 groups, the medium was added with urea 20 and 40 mg/dL, respectively. Each petri dish contained three drops of maturation medium (300 µl/drops) according to the groups. Microdrops were coated with mineral oil and then incubated in a 5% CO2 incubator, at 39 ˚C with maximum humidity. Aceto-orcein staining was conducted to evaluate the maturation of oocytes based on the achievement of metaphase II phase that is indicated by the presence of metaphase plate and/or first polar body. The result showed that the oocyte maturation rates of P0, P1, and P2 were 51.25, 52.43 (p >0.05), and 46.88 % (p <0.05) respectively. It could be concluded that the presence of urea at 40 mg/dL in maturation medium reduced the percentage of bovine oocyte maturation in vitro.

66 Effect of the co-culture system and the culture medium on invitro embryo development in alpacas (Vicugna pacos)

The aim was to evaluate 4 co-culture systems and 4 culture medium types to produce alpaca embryos by IVF. Gametes were obtained from ovaries and testes collected from a slaughterhouse. Oocytes were recovered by follicle aspiration using a 5-mL syringe. Oocytes with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured for 26h in an incubator (5% CO2 in air at 38.5°C), in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.02IU mL−1 FSH, 1µg mL−1 oestradiol 17β, 0.2mM sodium pyruvate, and 50µg mL−1 gentamicin sulphate. After maturation, oocytes were placed in FERT-TALP (fertilization- Tyrode’s medium with albumin, lactate, and pyruvate) solution for 30min before IVF with epididymal sperm. Motile sperm were selected by swim-up method. Oocytes were co-cultured with 3×106 spermatozoa mL−1 for 18 to 20h under the same atmospheric conditions mentioned above. In Experiment 1, we evaluated 4 co-culture systems: ear fibroblasts (T1, n=224), fetal fibroblasts (T2, n=154), granulosa cells (T3, n=225), oviduct cells (T4, n=169) and synthetic oviductal fluid (SOF) invitro culture (IVC) (T5, n=116). As culture base, we used 0.5mL of SOF IVC medium supplemented with 10% FCS in all treatments. In Experiment 2, we evaluated 4 culture media: TCM-199+10% FCS (T1, n=137), CR1aa+3mg of bovine serum albumin (BSA) mL−1 (T2, n=85), KSOM medium+3mg of BSA mL−1 (T3, n=110), and SOF IVC+10% FCS (T4, n=66). The volume of culture medium used was 0.5mL in 4-well Nunc plates for each treatment. The time (8 days) and culture (38.5°C, 5% CO2 in air and high humidity) conditions, and change of medium (each 24h) were the same in both experiments. Statistical significance was determined using ANOVA. The mean and s.d. were calculated from the average of the percentages obtained in each repetition. In experiment 1, the cleavage rates were higher (P&lt;0.05) in co-culture with fetal fibroblasts (46%±0.17), oviduct cells (50%±0.09), and ear fibroblasts (58%±0.17) than with granulosa cells (28%±0.12) and SOF IVC (30%±0.18). Also, the morula rates were higher (P&lt;0.05) in co-culture with fetal fibroblasts (35%±0.16), oviduct cells (31%±0.01), and ear fibroblasts (35%±0.14) than with granulosa cells (11%±0.01) and SOF IVC (27%±0.17). In contrast, there were no differences in blastocyst rates between co-culture with granulosa cells (10%±0.04), SOF IVC (12%±0.09), and fetal fibroblasts (24%±0.14). However, there were differences between co-culture with oviduct cells (19%±0.06) and ear fibroblasts (32%±0.14). In Experiment 2, there were no differences in cleavage rates between TCM-199+FCS (28%±0.12), CR1aa+BSA (44%±0.24), KSOM+BSA (38%±0.19), and SOF-IVC+FCS (50%±0.20). However, there were differences in morula rates between CR1aa+BSA (7%±0.09) and SOF IVC+FCS (35%±0.21), TCM-199+FCS (23%±0.09), and KSOM+BSA (27%±0.15). We obtained a higher blastocyst rates in SOF IVC+BSA (24±0.12) and KSOM+BSA than with CR1aa+BSA (3±0.06) and TCM-199+FCS (9±0.03). In conclusion, KSOM and SOF-IVC were the best media for culture, and oviduct cells and ear and fetal fibroblasts were the best cells to produce alpaca embryos by IVF.

The interrelationship between the occurrence of oversized follicles and the peripheral and intra-follicular concentrations of E2, P4, FSH, and LH in female dromedary camels

The present study aimed to clarify the phenomenon of presence of larger than normal follicles (OVGF) in female dromedary camels. Females with OVGF (n=125) were examined by manual palpation and ultrasonography. Accordingly, the OVGF were subdivided into those with thin walls and clear hypoechogenic content (OVGF-TH, n=18) and those with thick walls and fibrous trabeculae (OVGF-TK, n=107). Transvaginal follicle aspiration was performed in females with OVGF and from a control group with growing follicles (1-2 cm in diameter, GF group, n=5). Serum was collected at the same time of follicle aspiration and analyzed for Follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4) and estradiol-17β profiles (E2). The follicular fluid (FF) was analyzed for E2 and P4. The results showed that mean E2 concentration in FF and serum were lower in OVGF-TH and OVGHTK groups than in the GF group (P < 0.05). Difference between OVGF-TH and OVGH-TK groups was not significant. P4 in FF did not significantly differ among groups. Positive correlation was found between E2 in FF and E2 in serum (r = 0.495, r = 0.03). Mean FSH concentration in serum was higher in OVGF-TH and OVGH-TK groups than in the GF group (P = 0.03). Mean LH concentration was non-significantly (P=0.1) greater in OVGF-TH and OVGH-TK groups than in the GF group. In conclusion, female dromedary camels with OVGF had endocrine characteristics differed from camels with no OVGF. It seems that the high FSH and/or LH concentration(s) stimulated the continuing growth of the developing follicles to reach these large sizes, suggesting that the phenomenon of OVGF in camels is a pathological finding.

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 >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 >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 >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 < 0.05) 7 days after aspiration than those with <75% expanded COC. Calves in the 7-day FSH protocol had >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 < 0.05) and Day 5 (50.6 ± 28.0 ng mL−1 v. 4.5 ± 1.0 ng mL−1; P < 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 < 0.05) than calves in the 4-day FSH protocol, whereas no difference (P > 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.

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P < 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P > 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P < 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P > 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&M University.

Fertility Preservation in Female Patients with Breast Cancer – a Current Overview

Many premenopausal patients who develop breast cancer have not yet completed their family planning, so measures of fertility protection to preserve their fertile potential would be beneficial. Polychemotherapy causes irreversible damage to the ovarian follicles – irrespective of whether in a neoadjuvant or adjuvant setting – and this can sometimes result in permanent infertility. Depending on which cytostatic agents are used and on the age-related ovarian reserve of the woman, gonadotoxic risk must be classified as low, moderate or high. Options of fertility preservation include: a) cryopreservation of fertilised or unfertilised oocytes. After ovarian hyperstimulation, mature oocytes are retrieved by transvaginal follicle aspiration, after which they are cryopreserved, either unfertilised or on completion of IVF or ICSI treatment. During b) cryopreservation of ovarian tissue, about 50% of the ovarian cortex of one ovary is resected with the aid of a laparoscopic procedure and cryopreserved. The application of c) GnRH agonists as a medicinal therapy option is an attempt at endocrine ovarian suppression in order to protect oocytes, granulosa cells and theca cells from the cytotoxic effect of chemotherapy.