Transillumination increases oocyte recovery from ovaries collected at slaughter. A new technique report

An inexpensive and convenient method of collecting large number of oocytes for in vitro procedures is by aspiration of follicles visible on the surface of isolated ovary. This method yielded only moderate numbers of oocytes per ovary, and it was found that the yield could be improved by slicing the tissue to reach deep, cortical follicles. However, slicing was time consuming and increased chances for sepsis. We developed a new technique that allows direct viewing of cortical follicles for aspiration of oocytes by transillumination of the ovarian medulla and cortex with a Plexiglas rod inserted through a small incision at the hilus. The technique, called "Transillumination-Aspiration Ovary" (TAO), increased the oocyte yield by 50% per ovary. The oocytes are probably recovered from deeper follicles which are difficult to identify during regular oocyte aspiration. The oocytes had a normal grading and exhibited normal in vitro development efficiency. Using the "TAO" technique we recovered 777 oocytes from 2160 follicles in 106 ovaries, a recovery rate of 36% from follicles and a mean of 7.3 oocytes/ovary. When we aspirated only surface follicles, we obtained 523 oocytes in 1384 visible follicles in 107 ovaries, for a recovery rates of 37% but a mean yield of 4.9 oocytes per ovary. Mean number of follicles were 20.5% with TAO and 12.8% without, thus recovery rates of oocytes per follicle were similar with both methods, but yield of oocytes per ovary was higher with TAO, thus showing that the difference between the two methods lies in higher numbers of visible follicles with TAO. Moreover, with the TAO technique 71% of the total oocytes we recovered (n=551) were grade I or II oocytes, in which 52% cleaved to the 2 to 4-cell stage and 26% had reached the blastocyst stage. We conclude that the method is effective for accurately locating cortical and peripheral follicles that contain oocytes suitable for IVF and in vitro embryo production (IVP).     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.     

Maturation and fertilization of porcine oocytes from primordial follicles by a combination of xenografting and in vitro culture

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.     

Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes

Ability of ovarian oocytes from the domestic dog to complete nuclear maturation in vitro (IVM) varies markedly among donors and generally is 20% or less of all oocytes cultured. To identify the cause(s) underlying these significant variations in meiotic maturation (to metaphase II; MII), we retrospectively analyzed data from 1,643 oocytes recovered from 90 bitches for which stage of reproduction and season of year were known. Neither stage of reproduction (proestrus/estrus, diestrus, anestrus, or prepuberty) nor season (P > 0.05) influenced the ability of oocytes to achieve nuclear maturation in vitro. A second study was conducted to examine the impact of follicular size on meiotic maturation. Populations of large oocytes were recovered from four categories of follicles (ranging from <0.5 to > 2 mm in diameter) and cultured in TCM 199 for 48 hr. Follicular size influenced (P < 0.05) meiotic competence. Mean percentages of MII oocytes were 16.9 +/- 9.2, 26.1 +/- 7.6, 38.4 +/- 9.2, and 79.5 +/- 10.9 for oocytes recovered from < 0.5 mm, > or = 0.5-< 1 mm, 1-2 mm, and > 2 mm diameter follicles, respectively. In summary, stage of reproduction and season have no impact on the ability of dog oocytes to achieve nuclear maturation in vitro. However, we demonstrated for the first time that dog oocytes acquire meiotic competency during follicular development. IVM success of selected oocytes from large size follicles (almost 80%) is about 60% higher than measured in most previous studies involving randomly collected oocytes.     

Collection of oocytes through transvaginal ultrasound-guided aspiration of follicles in an Indian breed of cattle

The present study was undertaken in Karan Fries, an Indian breed of cattle to (1) determine the number of follicles available for puncture and (2) explore the potential of this breed as a donor of developmentally competent oocytes. Ovum pick-up (OPU) was performed using an ultrasound machine with a transvaginal convex transducer (5 MHz) with a needle guide, single lumen 19-gauge 60 cm long needle and a vacuum pressure of 90 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (>/=10 mm). The oocytes recovered were classified by quality. They were matured in vitro, irrespective of their grade, in 50 microl droplets of the in vitro maturation (IVM) medium (TCM-199+10% fetal bovine serum(FBS)+5 microg/ml follicle stimulating hormone (folltropin)+1 microg/ml estradiol-17beta+0.2 mM sodium pyruvate), covered with paraffin oil, in 35 mm petridish for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5 degrees C. The cleavage rate was recorded at day 2 post-insemination after subjecting the oocytes to in vitro fertilization (IVF). The differences in follicular populations of all size categories among individual donors were not significant. A total of 92 oocytes were recovered by aspiration of 157 follicles, with an overall recovery rate of 59% (range 35-79%). Of these, 32% were of grades A and B and the rest of grades C and D. The mean numbers of total follicles and the oocytes recovered per session did not differ significantly among individual donors. Out of the 73 oocytes subjected to IVM and IVF, 24 reached 2-4 cell stage at day 2 post-fertilization, with a cleavage rate of 33%. The total number of oocytes recovered was correlated with the number of small (R=0.54, P<0.01) but not with the number of medium and large follicles. This study demonstrates the use of OPU as a means of obtaining developmentally competent oocytes from an Indian breed of cattle for obtaining cattle oocytes in India where cow slaughter is not allowed for religious reasons.     

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence

The aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst-PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p < or = 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p < or = 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.     

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.     

Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.     

Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

Number and quality of oocytes in relation to age of cattle

An experiment was conducted to characterize the ovarian follicles as well as follicular oocytes in relation to cattle age. Ovaries from 67 heifers aged 18–24 months, 75 cows aged 3–8 years, and 70 cows aged 9–17 years were examined.In heifers 22.66 ± 12.56 (mean ± S.D.) 2–6-mm follicles were observed, from which 10.22 ± 6.17 oocytes were recovered; of these 4.63 ± 3.98 were morphologically normal. Similar values were observed in 3–8-year-old cows, i.e. 23.08 ± 13.95 follicles, 9.88 ± 6.24 oocytes, and 5.24 ± 4.03 normal oocytes. In cows aged >9 years 18.03 ± 10.83 (P < 0.05) follicles, 8.37 ± 5.87 oocytes and 3.89 ± 3.73 normal oocytes were obtained.No relationship was observed between the number of ovarian follicles and the quality of the recovered oocytes.     

Using hormone-treated pregnant cows as a potential source of oocytes for in vitro fertilization

In vivo collection of oocytes during pregnancy may be alternative method of obtaining gametes for in vitro fertilization (IVF) from genetically superior gestating cattle. The objectives of this experiment were to induce follicular growth in mature beef cows during each trimester of pregnancy, and then to collect oocytes and verify oocyte competency by IVF and subsequent embryo culture in vitro. Cyclic beef cows in Treatment A and pregnant cows in Treatment B were administered a total dose of 40 mg of FSH in descending dose levels (6, 5, 4, 3 and 2 mg) twice daily for 5 consecutive days. Cows in Treatment A were administered 25 mg of PGF(2)alpha and in Treatment B an equal volume of 0.9% saline at the seventh FSH injection. Pregnant cows in Treatment C were administered neither FSH nor PGF(2)alpha and served as a control group. Following a gonadotropin treatment, the ovaries of each female were evaluated for follicular development by ultrasonography. Oocytes were collected by follicle aspiration from cows in the first trimester. Following IVF procedures, the embryos were co-cultured on caprine oviductal cells, or in the chicken embryo co-culture system, or were placed in goat oviducts in vivo. The mean number of follicles per ovary 12 hours after FSH treatment was not different for cows in Treatments A and B, (8.1 vs 7.7) and both numbers were greater (P<0.05) than the 1.1 follicles per ovary for the control cows in Treatment C. Oocytes collected in vivo and exposed to IVF, resulted in 20% cleaving, and of these embryos 50% developed to the morula stage in culture. In summary, stimulating supplemental follicular development with FSH treatment during pregnancy and collecting the oocytes for IVF may be an alternative method for obtaining supplemental gametes from valuable donor cattle.     

In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH

The effect of treatment of donor cattle with progestagen and oestradiol or FSH on in vivo oocyte recovery and in vitro embryo production was studied. Forty-eight beefxFriesian cows formed eight replicates of six treatments in a 2 (no steroid versus steroid)x3 (none, single or multiple dose(s) of FSH) factorial design in which follicles were aspirated once weekly for 3 weeks. Oocytes were graded, washed, matured for 20-24h and then inseminated with frozen/thawed semen from a single sire followed by coculture on granulosa cell monolayers.Treatment with steroid had no significant effect on any follicular, oocyte or embryo production variate other than to reduce the number (P<0.05) and the diameter of large follicles>10mm (P<0.01) present at aspiration. FSH increased numbers of medium (6-10mm) and large follicles (P<0.01) and there was a corresponding decrease in the number of small follicles (2-5mm; P<0. 01). The total number of follicles at aspiration increased from 17. 7+/-1.60 for animals not treated with FSH to 23.6+/-1.97 following multiple dose treatment with FSH (P<0.05). Significantly, more follicles were aspirated following FSH treatment (no FSH 9.7+/-1.09, single dose FSH 13.6+/-1.30, multiple dose FSH 17.3+/-1.52; P<0.05) and numbers of oocytes recovered per cow per week increased (no FSH 4.1+/-0.76, single dose FSH 5.3+/-0.87, multiple dose FSH 5.9+/-0. 94) but the differences were not significant. Significantly, more good oocytes (Category 1) were recovered from animals treated with FSH (P<0.05). There was no overall significant effect of FSH on embryo production rate or the total number of transferable embryos produced but the number of transferable embryos was highest following administration of multiple doses of FSH.In conclusion, progestagen plus oestradiol 17beta treatment did not affect follicle, oocyte and embryo production of oocyte donors aspirated once per week. FSH treatment, however, significantly increased the number of follicles aspirated and Category 1 oocytes recovered. Multiple dose administration of FSH resulted in the production of the highest number of transferable embryos but this effect was not significant.     

Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production

Characteristics of the follicle population and oocyte developmental competence at selected stages of follicular development were studied in cows with the aim to increase embryo production derived from oocytes collected by transvaginal aspiration. In Experiment 1, the growth phase before dominant follicle selection and the low dominant phase during dominant follicle regression were compared. Twenty-four cyclic Holstein cows, 4 to 6 yr of age, were divided into 2 groups. Animals were synchronized using two injections of prostaglandin F2alpha at 11 d intervals, and onset of estrus was determined (Day 0). Using ultrasonography, all follicles were counted and classified. Oocytes were aspirated once on Days I through 3 (Group 1, n=5) or Days 15 and 16 (Group 2, n=3) of the estrus cycle. The experiment was carried out in 3 replicates. In Experiment 2, the growth phase of the first follicular wave before dominant follicle selection was characterized in detail. Twelve cows of the same breed and age were divided into 3 groups. Their first estrus was synchronized as in Experiment 1, and each following estrus was induced using one injection of prostaglandin F2alpha administered 4 to 6 d after each aspiration performed. The ovaries were examined, and oocytes were collected repeatedly (total of 5 times per cow) on Days 1 (Group 3, n=4), 2 (Group 4, n=4) or 3 (Group 5, n=4) after estrus at 10 d intervals during a 40 d period. Viable oocytes were matured, fertilized and cultured using the standard methods. In Experiment 1, the mean numbers (+/-SD) of all follicles and of recovered and viable oocytes per donor were higher in Group 1 than in Group 2, but only the mean numbers (+/-SD) of larger follicles and recovered oocytes were statistically significant (8.0 +/- 0.6 and 6.2 +/- 0.6.vs. 3.3 +/- 0.5 and 2.8 +/- 0.2; P< 0.05). In Experiment 2, the percentage of larger follicles out of all visible follicles and the mean numbers (+/-SD) of larger follicles per donor were significantly higher (P<0.05) in Groups 4 (75.7 and 9.1 +/- 2.7) and 5 (66.3 and 8.5 +/- 2.9) when compared to Group 3 (27.9 and 3.8 +/- 0.8). The development rate of fertilized oocytes was significantly higher (P<0.05) in Groups 4 (27.8) and 5 (27.5) than in Group 3 (12.8). It can be concluded that it is possible to improve the efficiency of transvaginal aspiration and in vitro embryo production by utilization of the growth phase of the first follicular wave before dominant follicle selection.     

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.     

Influence of follicular components on oocyte meiosis in vitro

Oocytes and follicular components obtained from ovaries recovered from mature Hereford cows at slaughter were used to determine follicular influence on oocyte maturation. Some oocytes were fixed immediately to determine the stage of maturation. The remaining oocytes were cultured for 32 to 34 hr in various environments to determine the influences of the granulosum and follicular fluids on meiotic changes. All noncultured oocytes had dictyate nuclei except one in premetaphase. Oocytes cultured in 50 or 100% follicular fluid or in contact with stratum granulosum cells showed some meiotic inhibition both before and after germinal vesicle breakdown (GVB). The least resumption of meiosis occurred in oocytes cultured in their intact follicles.     

The ability to achieve meiotic maturation in the dog oocyte is linked to glycolysis and glutamine oxidation

We tested the hypothesis that meiotic competence of dog oocytes is tightly linked with donor follicle size and energy metabolism. Oocytes were recovered from small (<1 mm diameter, n = 327), medium (1–<2 mm, n = 292) or large (≥2 mm, n = 102) follicles, cultured for 0, 24, or 48 hr, and then assessed for glycolysis, glucose oxidation, pyruvate uptake, glutamine oxidation, and nuclear status. More oocytes (P < 0.05) from large follicles (37%) reached the metaphase‐II (MII) stage than from the small group (11%), with the medium‐sized class being intermediate (18%; P > 0.05). Glycolytic rate increased (P < 0.05) as oocytes progressed from the germinal vesicle (GV) to MII stage. After 48 hr of culture, oocytes completing nuclear maturation had higher (P < 0.05) glycolytic rates than those arrested at earlier stages. GV oocytes recovered from large follicle oocytes had higher (P < 0.05) metabolism than those from smaller counterparts at culture onset. MII oocytes from large follicles oxidized more (P < 0.05) glutamine than the same stage gametes recovered from smaller counterparts. In summary, larger‐sized dog follicles contain a more metabolically active oocyte with a greater chance of achieving nuclear maturation in vitro. These findings demonstrate a significant role for energy metabolism in promoting dog oocyte maturation, information that will be useful for improving culture systems for rescuing intraovarian genetic material. Mol. Reprod. Dev. 79: 186–196, 2012. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.     

Repeated ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus bubalis): oocyte recovery and quality

The present study was undertaken to explore the potential of the Murrah breed of buffaloes as donors of oocytes and to find out the recovery rate and oocyte quality in cyclic Murrah buffaloes subjected to oocyte recovery once a week. Murrah buffaloes (n = 5) were synchronized for estrus by a single prostaglandin injection schedule. The animals were subjected to transvaginal oocyte retrieval (TVOR) once weekly for 6 weeks, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). TVOR was performed using an ultrasound machine with a 5 MHz transvaginal transducer, single lumen 19-gauge, 60 cm long needle and a constant vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (> or = 10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes (COCs) with > or = 5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. The mean (+/- S.E.M) number of small, medium and large follicles, and the number of total follicles observed per animal per session, which was 2.2 +/- 0.3, 0.6 +/- 0.2, 0.9 +/- 0.1 and 3.7 +/- 0.3, respectively, did not differ between animals or between puncture sessions. Small follicles constituted a major proportion (59%) of the total observed follicles. A mean (+/- S.E.M) number of 3.0 +/- 0.3 follicles were punctured and 2.0 +/- 0.3 oocytes recovered per animal per session, with a recovery rate of 68%. Out of the total 61 oocytes recovered, 36 (59%) were of grades A + B whereas 25 (41%) were of grades C + D. In conclusion, this study describes the potential of cyclic Murrah buffaloes as donors of oocytes collected by repeated TVOR once a week, without any adverse effects on follicular growth and oocyte recovery. It also describes an efficient system for carrying out TVOR in buffaloes.     

Ultrasound-guided follicle aspiration: effect of the frequency of a linear transvaginal probe on the collection of bovine oocytes

The effect of the frequency of an ultrasonic linear transvaginal probe on the collection of bovine oocytes by transvaginal ultrasound-guided follicle aspiration was investigated. Probes with different frequencies (7.5 or 5.0 MHz) were applied to examine the clarity of follicles on the monitor using ovaries of slaughtered cows in Experiment 1. The follicles were visualized on the monitor and divided into small (3- to 5-mm diameter) and large (6- to 10-mm) groups. They were also divided into 2 groups according to the clarity of their outline (clear or obscure). The number of small follicles visualized with a clear outline was greater (P < 0.01) with the 7.5 MHz probe than with the 5.0 MHz probe (9.0 vs 3.2). Oocyte aspiration from live cows was performed using the 7.5 or 5.0 MHz probe in Experiments 2 and 3. The recovered oocytes were divided into 3 categories: cumulus-oocyte-complexes (COCs), denuded oocytes and all others. In Experiment 2, the number of oocytes collected per donor cow was assessed, and in Experiment 3 the number of oocytes per aspirated follicle was examined by aspirating a constant number of follicles per aspiration session. The numbers of oocytes and COCs per donor cow obtained with the 7.5 MHz probe (11.2 and 9.0, respectively) were greater (P < 0.01) than those obtained with the 5.0 MHz probe (4.3 and 3.5). This difference between probes was due to the greater clarity of the follicle images obtained with the 7.5 MHz probe.