Aspiration Of Ovarian Follicles Research Articles

Fertility-sparing in cancer patients

Objective: This review aimed to investigate and summarize the current evidence on fertility-sparing options in cancer patients. Mechanism: Fertility preservation methods are becoming popular through the improved prognosis of cancer patients at a younger age and early diagnostic tools. However, currently, more and more women are suffering from iatrogenic ovarian failure and fertility loss because of cancer treatment. Most treatments have been used for hematological malignancies, but different gynecological cancers can be eligible. Findings in brief: Fertility preserving strategies such as oocyte and embryo cryopreservation, ovarian tissue preservation, ovarian transposition, and aspiration of ovarian follicles are the methods that could be suggested to the patients. The current knowledge supports oocyte and embryo cryopreservation as feasible, safe, and effective treatment approaches for cancer patients seeking fertility preservation. Conclusions: Robust evidence is still needed to prove the effectiveness of cryopreservation of the ovarian tissue and ovarian follicle aspiration approaches since these techniques are still in early their steps.

12 Artificial insemination and embryo transfer results in ewes during a long daylength period

Sheep are polyestrous, short-day breeders (∼11h of daylength), and exhibit oestrus approximately every 16–17 days during the breeding season, usually in late September to late December in the Northern Hemisphere. Progressive sheep producers often use assisted reproductive techniques such as laparoscopic AI and ovarian hyper-stimulation, embryo collection, and embryo transfer (ET) to increase genetic gain, and strive to have early December lambs to target specific show markets, which dictates AI or ET during the late summer. This field trial compared pregnancy rates following AI or ET in July and August (∼14h of daylength) in southwest Virginia (36–38′12″ N). Ewes (AI, n=83; ET recipients, n=33) were synchronized using a modified Ovsynch protocol involving intravaginal progesterone implants for 14 days, prostaglandin F2α (intramuscular) 48h before expected oestrus, and PG600 (IM) and gonadotrophin-releasing hormone (intramuscular) 52 to 54h and 16h before AI, respectively. Ewes were subjected to AI (frozen/thawed semen) regardless of whether they displayed signs of oestrus, and ewes selected as embryo recipients were subjected to a similar protocol but instead received 1 or 2 embryos (based upon the number of viable embryos produced per embryo donor) 6 days following the AI of the embryo donors. Ovarian hyper-stimulation of the embryo donors (n=13) was enabled by twice-daily FSH injections [totalling 350–455IU of Folltropin V (10–13mL)] for the 4 days before AI. Six days following AI, embryos were recovered surgically from the embryo donors (n=13) and yielded an average (±s.e.m.) of 6.6±1.2 total ova, 4.7±1.1 transferable quality embryos, and 1.9±0.8 unfertilized ova per collection. Pregnancy was detected using transrectal ultrasonography at ∼30 days of gestation and the pregnancy rates were analysed using Chi-squared. There was a tendency (P=0.092) for more pregnancies to be established following ET (22/33; 66%) compared with AI (41/83; 49%). There was no statistical relationship between AI ewes or ET recipient ewes that became pregnant relative to whether they displayed signs of oestrus or not. Embryo transfer was a more successful approach to produce pregnancies in ewes compared with AI during long daylength periods in this field trial. Further, ova from hyper-stimulated embryo donor ewes experienced a very high fertilization rate. Future studies will evaluate the ova capability directly via laparoscopic aspiration of ovarian follicles and IVF and evaluation of hyper- and non-hyper-stimulated follicular waves (using transrectal ultrasonography) and endocrine dynamics during different long and short daylength periods. Extending the opportunity to generate embryos and offspring regardless of daylength will assist aggressive sheep producers in reaching their reproductive, financial, and genetic goals.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

158 FIRST LLAMA BORN BY IN VITRO FERTILIZATION

The aim was to transfer alpaca and llama embryos obtained by IVF into recipient llamas and evaluate pregnancy and birth rates. Gametes were collected from samples of ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered by aspiration of ovarian follicles using a 5-mL syringe, where oocytes with at least 3 layers of cumulus cells and a homogenous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.1 IU), and oestradiol 17β for 30 and 36 h with 5% CO2 in air, for alpaca and llama, respectively. After the maturation time, COC were placed in FERT-TALP solution 30 min before in vitro insemination with epididymal sperm of alpaca and llama as appropriate, the sperm were selected by swim-up method with centrifuging twice in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-free BSA, in an incubator with 5% CO2 in air set at 39°C for 45 min, oocytes were co-incubated with sperm concentration of 3 × 106 mL−1 for 18 to 20 h at 39°C with 5% CO2 in air. The IVF was carried out the day of ovulation induction of recipients. A group of 15 morphologically intact Day-8 blastocysts derived from in vitro culture with SOF serum were transferred nonsurgically into the uterine horn ipsilateral to the corpus luteum of 15 synchronized recipient llamas with an intravaginal device (controlled internal drug release) for 8 days. Then, 6 days after controlled internal drug release removal, ovulation was induced in recipients with the application of 1 mL of GnRH with previous ultrasound confirmation of the presence of a dominant follicle greater than 7 mm in diameter. Nine alpaca blastocysts and 6 llama blastocysts were transferred. The pregnancy rate was assessed by ultrasound at 45 days after transfer. The results obtained were as follows: for pregnancy rate, 33.3% (3/9) and 50% (3/6) for alpaca and llama embryos, respectively; for birth rate, 0.0% (0/9) and 16.7% (1/6) for alpaca and llama embryos, respectively. An alpaca fetus and 2 llama fetuses were aborted between 7 and 10 months of pregnancy, and only a llama gestation ended successfully, producing the first birth of the world of a llama bred by IVF in South American camelids, demonstrating that it is possible to obtain viable offspring in these species using this biotechnology.

199 EFFECT OF UROKINASE TYPE PLASMINOGEN ACTIVATOR DURING IN VITRO MATURATION OF BOVINE OOCYTES AND EARLY EMBRYO PRODUCTION

The study of molecules involved in the oocyte maturation and early embryo development is crucial to improve the conditions of in vitro embryo production. The plasminogen activation system is involved in the initial steps of reproduction and urokinase type plasminogen activator (uPA) is expressed in the granulosa cells (GC) of bovine cumulus-oocyte complexes (COC). The aim of this study was to analyse the effect of uPA in bovine oocyte in vitro maturation (IVM). We have analysed whether the addition of uPA or the inhibition of its proteolytic activity affects IVM. We have evaluated (1) nuclear and cytoplasmic maturation, (2) developmental competence, and (3) oocyte and GC gene expression. Immature COC were obtained by aspiration of ovarian follicles of slaughtered heifers. Selected COC were in vitro-matured in 4 groups: control, 10 nM uPA, dimethyl sulfoxide control, and 100 μg mL–1 amiloride, a specific inhibitor of uPA proteolytic activity (4 replicates). After 24 h of IVM, oocytes of each treatment were either fixed and stained with Hoëscht (to evaluate nuclear maturation) or LCA-FITC to analyse the cortical granules distribution as a marker of cytoplasmic maturation (n = 10/group per treatment per replicate). In addition, pools of 10 oocytes and their separated GC were snap-frozen to analyse by qRT-PCR their profile expression of genes related with apoptosis (BAX, BCL2, TP53, SHC1), cell junctions (GJA1, TJP1), cell cycle (CCNB1), oxidative stress (SOD2, GPX1), oocyte quality (BMP15, GDF9), and serpin proteases inhibitors (SRP1, SRP5), normalised respect 2 housekeeping genes (H2AFZ, ACTB). The remaining COC were fertilised (Day 0) and in vitro cultured to evaluate developmental competence in terms of cleavage rate (Day 2) and blastocyst yield (Days 7–9). All data were analysed by one-way ANOVA. In the presence of amiloride, a significant reduction in the oocyte maturation was observed at both levels; 83.33% of oocytes remained in vesicle stage, and 75.0% showed a cortical granules distribution of type I. The rest of the groups (62.67%, 62.65%, and 60.29%) reached metaphase II (MII), and 51.66%, 32.9%, and 25.44% showed granule distribution of type III. For embryo development, the amiloride group had a cleavage rate and blastocyst yield significantly lower compared with controls (23.23% v. 80.85% and 83.83%; 4.45% v. 25.21% and 25.21%, respectively), whereas uPA treatment had no effect (84.28% and 24.16%). In presence of amiloride, the transcript levels of TJP1, GJA1, and CCNB1 were up-regulated, whereas SOD2, SRP1, and SRP5 were down-regulated in GC compared with all other groups (P < 0.05). No differences were observed in oocytes gene expression between treatments. In conclusion, although the addition of exogenous uPA does not alter oocyte maturation, the specific inhibition of the proteolytic activity of uPA by amiloride reduced IVM of bovine oocytes and altered the expression of genes related to cell junctions, cell cycle, oxidative stress, and serpins of GC, indicating that proteolytic activity of uPA is critical for oocyte IVM in bovines.

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

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

133 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON THE DEVELOPMENT OF IN VITRO-MATURED-IN VITRO-FERTILIZED-IN VITRO-CULTURED BOVINE EMBRYOS

The objective of this study was to investigate whether progesterone (P4) supplementation to in vitro maturation (IVM) medium could affect the competence of bovine oocyte to develop into blastocysts in vitro. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (2 to 6 mm in diameter) obtained from a local abattoir. The COC were matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 FSH at 38.5° in an atmosphere of 5% CO2 in air. After 18 h of gamete co-culture (5 × 106 sperms mL–1), presumptive zygotes were cultured in CRlaa containing 5% calf serum at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days (fertilization = Day 0). Progesterone was added to the IVM medium 10 h after the start of the culture (1 μg group = 1 μg mL–1 of P4; 5 μg group = 5 μg mL–1 of P4; control group = no P4). The maturation (MII) rates were investigated after 20 h of starting the IVM culture. After maturation, the COC were denuded mechanically, and a part of the oocytes were mounted on slides, fixed with aceto-alcohol (1 : 3) solution for 48 h, stained with aceto-orcein, and observed under a phase-contrast microscope to determine their nuclear status (1 μg group: n = 32; 5 μg group: n = 28; control group: n = 31). The remaining COC were used for IVF. The cleavage rates were investigated on day 2, and the blastocyst formation rates were investigated on Days 7 to 9, respectively (1 μg group: n = 264; 5 μg group: n = 274; control group: n = 277). The blastocysts from Day 7 were used for differential staining of the inner cell mass (ICM) and trophectoderm cells (TE). The total cell numbers, ICM, and TE in the blastocysts were counted (1 μg group: n = 28; 5 μg group: n = 24; control group: n = 24). The rates of MII, cleavage, and blastocyst formation were expressed and analysed by the chi-squared test. Each set of cell numbers (mean ± standard error) was analysed by the unpaired t-test. The MII rate in the control group (76.7%) was significantly lower (P < 0.05) than that in the 1 μg group (93.8%). The cleavage rate in the 1 μg group (85.6%) was significantly higher (P < 0.05) than those in the control group (74.7%) and 5 μg group (77.4%). Further, the blastocyst formation rate in the 1 μg group (47.7%) and 5 μg group (43.4%) were significantly higher (P < 0.05) than that in the control group (35.0%). The ICM numbers (mean ± s.e.) were 39.5 ± 13.8 to 36.2 ± 8.9, the TE numbers were 74.4 ± 22.4 to 66.2 ± 12.9, the total cell numbers of blastocysts were 110.6 ± 28.2 to 103.0 ± 13.8. There was no significant difference in cell numbers among the groups. These results indicate that the cleavage and blastocyst formation rates can be improved by the addition of 1 μg mL–1 of P4 to the maturation medium.

Application of Reproductive Biotechnology for the Recovery of Endangered Breeds: Birth of the First Calf of Murciana–Levantina Bovine Breed Derived by OPU, In vitro Production and Embryo Vitrification

In a conservation project, reproductive biotechnology was implemented for the recovery and conservation of an endangered bovine breed in Spain. The breed Murciana-Levantina, declared to be worthy of special protection status (http://www.fao.org/newsroom/en/news/2006/1000464/index.html), is of great interest because of its hardness, longevity, docility and disease resistance. This contribution describes the birth of the first calf of this breed obtained by reproductive biotechnology, using ultrasound-guided punction and aspiration of ovarian follicles, in vitro embryo production, vitrification of embryos by a cryotop device and, finally, the transfer of cryopreserved embryos to recipient heifers of a commercial dairy herd.

144 EXPRESSION OF ACETYL COENZYME A CARBOXYLASE α (ACCα) IN FELINE, CANINE AND PORCINE OOCYTES

High lipid content in in vitro–derived embryos of several species is associated with poor developmental potential and cryosurvival of oocytes. Lipid content of oocytes varies among species and embryonic lipid content is further influenced by the culture environment. In spite of the variation among species, the relative contribution of de novo lipid synthesis during oocyte maturation or subsequent embryonic development is unknown. In the present study, we evaluated the expression of acetyl coenzyme A carboxylase (ACCα), the key rate-limiting enzyme of lipogenesis, in oocytes of 3 species with high lipid content. Ovaries of dogs and cats were collected from a local veterinary clinic and those of pigs from an abattoir. Cat and dog oocytes were recovered by the slicing method of the ovaries. Porcine oocytes were recovered by aspiration of ovarian follicles. Immediately after collection, oocytes were fixed for 30 min using a solution of 10% neutral buffered formalin. The ACCα enzyme expression was evaluated in porcine, cat and dog oocytes by immunofluorescence using a goat anti-human ACCα primary antibody followed by fluorescein isothiocyanate-conjugated anti-goat secondary antibody. The study was replicated, with negative controls, 3 times using ≥30 oocytes per species. Messenger RNA expression of ACCα gene was also evaluated in pig oocytes. The RNA was isolated from fat tissue, pooled good-quality oocytes (n = 30), pooled poor-quality oocytes (n = 30) and cumulus cells using the RNeasy Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Complementary DNA was synthesised from 200 ng of RNA using Quantitect reverse-transcription kit (Qiagen) according to the manufacturer's instructions. Real-time PCR assays were carried out in duplicate and expression of ACCα mRNA levels relative to fat was determined. Oocytes and cumulus investment in cats, dogs and pigs strongly expressed ACCα. Expression of the protein was uniformly distributed through the entire ooplasm. The mRNA expression of ACCα in good- and poor-quality oocytes and cumulus cells relative to fat tissue was 11.5, 1.4 and 40.1%, respectively. Further studies are warranted on the dynamics of expression of ACC during in vitro maturation and the functional activity of the enzyme. We extend our appreciation to the Alabama Animal Alliance Inc. (Montgomery) for providing us with dog and cat ovaries; the Lambert-Powell Meats Laboratory of Auburn University for donating pig ovaries; and Dr. James Webster, Dr. Tsegaye Habtemariam and Dr. Abdalla Eljack for administrative support.

166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS

An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P < 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P < 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems. Table 1.Effect of different culture methods for bovine embryo development

In Vitro and In Vivo Developmental Potential of Nuclear Transfer Embryos Using Bovine Cumulus Cells Prepared in Four Different Conditions

We examined the effect of culture of donor cells on nuclear transfer efficiency using bovine cumulus cells treated with four different conditions: (1). group A, the cells removed from cumulus-oocyte complexes (COC) after aspiration of ovarian follicles; (2). group B, the cells removed from COC after in vitro maturation; (3). group C, the cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (4). group D, the cells cultured in DMEM with 0.5% FBS for an additional 5 days. Analysis of cell cycle using flow cytometry revealed that the relative proportion of donor cells at G0/G1 phase of cell cycle was 89.7% in group A, 89.5% in group B, 76.0% in group C, and 90.6% in group D. The developmental rates to blastocyst stage in groups C (45.3%) and D (46.4%) were significantly (p < 0.05) higher than in groups A (17.5%) and B (31.9%). After transfer of blastocysts produced in each group, nine of 24 recipients became pregnant on day 30. A total of five live calves were obtained from cumulus cells in all groups (group A [n = 1], group B [n = 1], group C [n = 2], and group D [n = 1]).

Blood loss following noncomplicated transvaginal oocyte retrieval for in vitro fertilization

Blood loss following noncomplicated transvaginal oocyte retrieval for in vitro fertilization

Ovarian follicular development following administration of progesterone or aspiration of ovarian follicles in holstein cows

The objective of this study was to compare the effects of administration of a single injection of progesterone (P 4) and follicle aspiration on Day 7 of the estrous cycle on the timing and synchrony of follicular wave emergence, time of ovulation, and concentrations of P 4, estradiol and FSH in Holstein cows. Twenty cows were assigned to 4 groups (n=5 cows per group) in a 2 by 2 factorial arrangement. Cows were treated on Day 7 (Day 0=estrus) of the estrous cycle with either sham follicular aspiration and an oil vehicle administered intramuscularly (control), aspiration of ovarian follicles (aspiration), 200 mg of P 4 im, or aspiration and 200 mg of P 4 im (aspiration + P 4). On Day 11, PGF 2α(25mg) was administered to all groups. Synchrony of ovulation was less variable in each of the treatment groups compared with the control group (P<0.05), whereas ovulation was delayed in cows in the P 4 group (P<0.05). Day of follicular wave emergence was delayed in the cows of the P 4 group compared with cows in the aspiration and aspiration + P 4 groups (P<0.01), whereas variability in wave emergence was less among both groups of aspirated cows compared with the cows in the control group (P<0.01). More follicles 4 to 7 mm in diameter were detected in the 2 aspiration groups compared with the cows in the control and P 4 group (P<0.05). No difference was detected among groups in the maximum concentration of FSH associated with follicular wave emergence. We conclude that both the administration of P 4 and the aspiration of follicles on Day 7 of the estrous cycle improves the synchrony of ovulation when luteolysis is induced on Day 11 and results in similar concentrations of FSH at the time of follicular wave emergence, but the timing of wave emergence and the number of follicles post-emergence differ.

Role of intravenous albumin in the prevention of severe ovarian hyperstimulation syndrome.

IntroductionOvarian hyperstimulation syndrome (OHSS) is a potentiallylife-threatening complication of ovulation induction, with anoverall incidence of 23.3% (Golan et al., 1989). Despite manyyears of clinical experience, there is no reliable test whichpredicts patients who will subsequently develop OHSS, thepathophysiology of this syndrome is not well understood, andno specific treatment is currently available. Many therapeuticoptions have been suggested in attempts to ameliorate andreduce the risk of severe OHSS (Orvieto and Ben-Rafael,1997). These include: reduction of the ovulatory dose of humanchorionic gonadotrophin (HCG) or withholding of HCG withcycle cancellation; substitution of HCG with gonadotrophin-releasinghormoneoritsagonist;conversiontoin-vitrofertiliza-tion (IVF) cycle; use of progesterone for luteal support insteadof HCG; cryopreservation of all embryos to avoid endogenouspregnancy-derived HCG; and repeated aspiration of ovarianfollicles and early corpus luteum.In 1993, Asch et al. proposed the use of i.v. administrationof human albumin solution during and immediately afteroocyte retrieval as a safe, effective, and economical treatmentfor the prevention of severe OHSS in high-risk patients. Sincethat time, several contradictory reports have emerged. The aimof the present study was to comprehensively review the dataon the role of i.v. albumin in the prevention of severe OHSS.Prevention of a diseaseTerminologyThe term ‘prevention’ refers to the acts of averting theoccurrence of disease (primary prevention) and of reversingor retarding the disease process before it has become clinicallyapparent (secondary prevention). Minimizing the effects of analready clinically apparent disease is considered treatment(tertiary prevention) (Orvieto et al., 1993).Intravenous albumin in the prevention of severe OHSSSupportive studies (Table I)Asch et al. (1993) administered 50 g of human albumin i.v.to 36 women considered at risk of severe OHSS by oestradiol3306

In vitro fertilization of cattle and sheep follicular oocytes by goat spermatozoa

The study compared efficiency of goat spermatozoa to penetrate cattle and sheep intact follicular oocytes, and evaluated effect of cumulus cells on the ability of sperm to penetrate heterologous oocytes. Ruminant cumulus-oocyte complexes (COC) were obtained by aspiration of ovarian follicles and were matured by culturing them in medium 199 plus goat serum in microdrops under paraffin oil for 24 h. Fresh semen was collected by artificial vagina, incubated for 4 h at room temperature, washed, and spermatozoa were then suspended in Talp medium plus goat serum and treated for capacitation by using incubation at 39°C and heparin. In the first experiment, goat, cattle and sheep matured oocytes were incubated in goat sperm preparation, whereas in the second experiment, cattle and sheep oocytes either partially (cumulus enclosed oocytes = + ) or totally denuded from granulosa cells (denuded oocytes = − ) were added to the prepared spermatozoa. Fertilization rates obtained in the first experiment (goat sperm-goat oocytes: 91.3%; goat-cattle: 82.7%; and goat-sheep: 79.8%, P > 0.05), showed that goat sperm were highly efficient in penetrating cattle and sheep oocytes as compared with homologous fertilization, and that goat sperm penetrated equally well cattle and sheep oocytes: Fertilization rates from the second experiment (cattle oocytes: +74.0%, −88.89%; sheep oocytes: +68.42%, −78.85%, P > 0.05), showed that cumulus cells did not modify the ability of goat spermatozoa to penetrate heterologous oocytes.