Effects of needle tip bevel and aspiration procedure on the morphology and developmental capacity of bovine compact cumulus oocyte complexes

Effects of the needle tip bevel and the aspiration procedure on the morphology of cumulusoocyte-complexes (COCs) and the developmental capacity of the oocytes after IVF were studied in 2 in vitro oocyte pick-up (OPU) simulations using a disposable ovum pick-up needle guidance system. In Experiment 1, the influence of the length of the needle bevel was investigated using a short and a long bevelled 20-g disposable needle. After being aspirated from slaughterhouse ovaries, the retrieved COCs were divided into 3 categories: 1) oocytes surrounded by a compact cumulus, 2) oocytes with an expanded cumulus, 3) partially naked oocytes. In Experiment 2, the influence of 5 different levels of aspiration vacuum for 3 different needle diameters (18-g, 19-g, 20-g) and 2 different needle bevels (long, short) was tested on the recovery and on the morphology of the cumulus investment of a fixed number of previously scored compact cumulus oocytes complexes (CCOCs), retrieved after slicing slaughterhouse ovaries. The re-retrieved COCs were allocated to Categories 1 and 3. The results show that the length of the needle bevel has a significant effect on oocyte recovery, in favor of the long-bevelled needle. As soon as higher aspiration vacua are used, a decrease of the number of CCOCs can be observed, which is less prominent for the short-bevelled needle compared to the long-bevelled one. The final number of blastocysts is similar for both needle types. In Experiment 2, the disposable needle system proved to be highly effective since nearly 80% of the CCOCs were retrieved. At low aspiration vacuum, up to 90% of the CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs, which is less pronounced using thinner needles. Averaged over all needle types, the prevalence of blastocysts expressed relative to the number of recovered oocytes decreases with higher aspiration vacuum.     

The collection of oocytes from bovine ovaries

Various needle sizes (17- and 20-g) and aspiration pressures (25, 50, 75 and 100 mmHg) were used to aspirate a total of 5,827 ovarian follicles from bovine ovaries from a slaughterhouse source to assess the impact on the quantity and quality of recovered immature oocytes. The cumulus oocyte complexes (COC's) were graded according to the presence and consistency of cumulus cells surrounding the oocyte and the data analyzed using general linear models. Overall recovery rates and the recovery of oocytes considered viable for IVM/IVF procedures (Classes A, B and C) were both significantly higher using a 17-g needle than a 20-g needle (P < 0.01). As the vacuum pressure increased so did the recovery rate of the total number of oocytes, although the number of viable oocytes reached a maximum at a calculated vacuum pressure of 55 mmHg for the 17-g needle and 77 mmHg for the 20-g needle, with an increased incidence of denuded oocytes at higher vacuum pressures. In a second experiment conducted on 1, 473 follicles, no significant difference was found between 17-g double (flushing) and 17-g single lumen needles in the recovery rate of either the total number or number of viable oocytes when using a vacuum pressure of 50 mmHg.     

Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes

The effects of aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complex (COC) and developmental capacity of the oocyte after IVF was studied in 2 experiments using a disposable ovum pick-up needle guidance system whose construction permits its use in vitro. In Experiment 1, the relationship was determined between the aspiration vacuum, expressed in millimetre of mercury, and the actual amount of water aspirated by the system, expressed in millilitre per minute. In Experiment 2, five different levels of aspiration vacuum for 3 different needle diameters (18g, 19g and 21g) were tested in slaughterhouse ovaries. The cumulus-oocyte complexes (COCs) were divided into 3 categories: 1) oocytes with a compact cumulus, 2) oocytes with an expanded cumulus and 3) naked oocytes. The results show that a change of needle diameter can triple the amount of fluid actually aspirated. The highest oocyte recovery rates are obtained when using the thickest needle (18-g), regardless of the aspiration vacuum. On the average, for all needle types, more oocytes are recovered at the highest aspiration vacuum. For all needle diameters, the proportion of oocytes surrounded by a compact cumulus decreases progressively as the vacuum increases. Regardless of the vacuum applied, thinner needles result in a higher proportion of recovered COCs with a compact cumulus. At a high aspiration vacuum, naked oocytes become predominant regardless of the needle diameter. The prevalence of blastocysts, expressed in proportion to the recovered COCs, decreases as the aspiration vacuum increases, being especially noticeable between 70 and 130 mm Hg.     

Factors affecting follicular population, oocyte yield and quality in camels (Camelus dromedarius) ovary with special reference to maturation time in vitro

Three experiments were conducted to study a series of factors affecting in vitro reproductive parameters in camels. In Experiment 1, the effect of season and presence of a corpus luteum (CL) on ovarian follicular populations, oocyte yield and quality was studied using a total of 252 and 208 ovaries collected during the breeding and non-breeding season, respectively. Small, medium, large and the total number of ovarian follicles, oocyte yield and quality were measured. In Experiment 2, the effect of methods of oocyte retrieval and needle gauge on oocyte yield and quality was evaluated with oocytes recovered using slicing and aspiration with 18-, 19- or 20-gauge needle. Oocytes were evaluated microscopically and classified into three categories. The objective of Experiment 3 was to identify the optimum time for oocyte maturation in the dromedary camel. Oocytes were cultured in CR1aa medium at 38.5 degrees C under 5% CO(2) for 24, 32, 36, 48 and 72h. Maturation was calculated as the percentage of cumulus expansion and oocytes reaching metaphase II (MII). The number of small, medium, large and the total number of ovarian follicles were higher (P<0.01) during the breeding than non-breeding season. The recovery of total number of oocytes and Category I oocytes were also greater (P<0.01) during the breeding season. Ovaries without a CL possessed significantly (P<0.01) more ovarian follicles and more (P<0.05) small and large follicles. The total number of oocytes and Category I oocytes were also greater (P<0.01) in ovaries without CL. Slicing of camel ovaries increased (P<0.01) the yield of oocytes as compared to aspiration. The aspiration of follicles using a 20-gauge needle had greater yields of the total number of oocytes and Category I oocytes than when using 19- (P<0.05) and 18-gauge needle (P<0.01). The culture of camel oocytes for 36h produced higher (P<0.01) percentages of cumulus expansion and oocytes at MII. Increasing culture times up to 48 or 72h increased (P<0.01) the percentage of degenerated oocytes.In conclusion, the growth and development of ovarian follicles in the camel as well as yields of Category I oocyte were greater during the breeding season. Slicing or aspirations using a 20-gauge needle yielded greater numbers of total and Category I oocytes. Finally, maturation of oocytes in CR1aa medium for 36h produced higher percentages of cumulus expansion and oocytes at MII stage.     

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.     

Methods for collecting follicular oocytes from mares

A series of experiments was conducted to develop a procedure for consistent, repeatable collection of oocytes from the preovulatory follicle of the mare. In one experiment, in situ follicular aspiration with a needle and syringe was performed on 19 mares. From 37 aspirations, four oocytes were recovered (10% recovery rate). In a second experiment, ovaries were visualized via standing flank laparotomy during which two different aspiration techniques were used. Use of a needle and syringe as in the first experiment resulted in successful oocyte recovery in one of seven (14%) attempts. Aspiration via a continuous irrigation vacuum system (CIV), developed for use during laparotomy, resulted in collection of oocytes from six of 10 (60%) attempts. In the third experiment, oocytes were recovered from seven of 18 (38%) attempts at in situ follicular aspiration using a double-lumen needle attached to the CIV. In each experiment, some mares were subjected to stimulation of follicular maturation by exogenous hormones. Oocyte recovery was significantly increased in treated mares as compared with nontreated mares. Results indicate that collection of equine follicular oocytes by in situ aspiration is possible with moderate success. Oocytes apparently are not physically damaged by the procedure, as most retained either the corona radiata or the entire cumulus cell mass.     

The effect of harvesting technique on efficiency of oocyte collection and different maturation media on the nuclear maturation of oocytes in camels (Camelus dromedarius)

The purpose of this investigation was to develop an efficient method for harvesting oocytes from dromedary camel ovaries and to examine the effect of different maturation media on their subsequent maturation in vitro. Oocytes were collected by aspirating the follicular contents using a needle attached to a syringe (Method I, n=163 ovaries) or to a constant aspirating pressure, applied by a vacuum pump (Method II, n=117 ovaries). Individual follicles were excised from ovaries and follicles were punctured with two needles (Method III, n=117). Oocytes were matured in vitro for 40-42 h. At the end of maturation period, oocytes were denuded of cumulus cells and the proportion of oocytes in metaphase-II (MII) stage was determined. In the second experiment, oocytes collected by the dissection method were matured in Tissue Culture Medium199 (TCM), CR1 or modified Connaught Medical Research Laboratories medium-1066 (CMRL) and their nuclear maturation was evaluated after 40-42 h. The recovery rate of oocytes was higher (P<0.01) with Method III compared with Method I or II (94, 31 and 33%, respectively). A higher proportions of oocytes collected with Method I or II were either completely or partially denuded compared with Method III (31, 14% versus 1%). The proportions of viable oocytes (78, 60 and 70%, respectively) and those showing metaphase II was not different (39, 50 and 46%, respectively, P>0.05) among the three treatment groups. Oocyte maturation rate was higher (P<0.05) when TCM was used compared with CMRL or CR1 medium. There was, however, no difference in the maturation rate for oocytes cultured in CMRL or CR1 medium. It may be concluded that a higher proportion of cumulus enclosed oocytes may be recovered by follicle dissection method compared to aspiration using syringe or pump. The higher recovery rate with a comparable proportion of viable and matured oocytes resulted in the overall increase in the number of matured (MII) oocytes/ovary with follicle dissection procedure compared with aspiration procedures. For in vitro maturation of oocytes, TCM is superior to CR1 and CMRL as basic maturation medium for this species.     

Meiosis in Bovine Oocytes Matured in Vitro and in Vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.     

Improved collection and developmental competence of immature macaque oocytes

Methods previously described to aspirate immature oocytes from ovaries of macaques result in approximately half the oocytes being stripped of cumulus cells. Here, we describe modifications of the needle aspiration assembly that yield much higher percentages of cumulus-intact oocytes when used with an ultrasound-guided method for oocyte recovery in monkeys. Sealing of the needle assembly appears to stabilize vacuum pressure at the needle tip and prevents air from entering the tubing. Reduction of the vacuum pressure from -100 to -20 kPa resulted in a significant decrease of denuded oocytes from over 50% to fewer than 10%. This was accompanied by a significant increase in the percentage of oocytes that developed into blastocysts after in vitro fertilization. Reduction of the aspiration pressure below -20 kPa significantly reduced the total number of oocytes recovered. We concluded that these modifications represent the best compromise to collect the largest number of cumulus-intact oocyte complexes from macaques.     

Endoscopy in cattle by the paralumbar route: Technique for ovarian examination and follicular aspiration

The ovary and follicular aspiration, or both, were observed in 50 heifers by endoscopy via the right paralumbar fossa. A total of 129 laparoscopies were performed. Eight animals underwent more than four interventions. Occasionally adhesions were observed, but they never interfered with ovary examination and follicular aspiration. The rate of ovum recovery was higher when a suction device was used rather than a syringe system (P < 0.01). The bore size of the needles influenced the proportion of oocytes obtained from the follicular aspirates. The use of a 19-G (1.00 mm) needle with a 45-degree bevel and a vacuum pressure of 250 mmHg gave the best results (recovery rate: 72%). This was significantly higher (P < 0.01) than the results obtained with a 21-G needle and a vacuum pressure of 500 mmHg (recovery rate: 53%).     

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.     

In vitro fertilization and development of bovine oocytes recovered from the ovaries of individual donors: A comparison between the cutting and aspiration method

Bovine oocytes were recovered from ovaries by either the cutting or the aspiration method, after which the oocytes were fertilized and cultured in vitro to investigate their developmental ability. In the cutting method, the surface and the interior of ovaries were cut with a set of 10 razors stacked at 2-mm intervals in modified TCM-199 medium supplemented with 5% fetal bovine serum; the liberated oocytes were then collected. In the aspiration method all visible follicles (2 to 5 mm in diameter) at the ovarian surface were aspirated with a syringe and an 18-gauge needle. Significantly more oocytes were recovered by the cutting than the aspiration method (mean: 63.3 vs 22.1), and the proportion of Rank A oocytes was also higher for the cutting method (84.6 vs 41.3%). Although no significant differences were observed between the 2 methods in the proportion of fertilized oocytes developing to the blastocyst stage in culture, the average number of blastocysts obtained by the cutting method was about 3.6-fold higher than by aspiration. The blastocysts were transferred nonsurgically to 37 (cutting method) and 36 (aspiration method) recipients, and 22 (59.0%) and 19 (52.8%), respectively, became pregnant.     

Efficacy of linear and convex transducers for ultrasound-guided transvaginal follicle aspiration

A new device was developed to hold linear transducers for transvaginal follicle aspiration. Efficacy of follicle aspiration was compared using a linear 6 MHz and a convex 5 MHz transducer. Fifty-five cows were submitted to follicle aspiration at random days of the estrous cycle. Aspirations were conducted with linear (n=28) and convex (n=38) transducers with 18 G needles at a negative pressure corresponding to 13 ml H(2)O/min. A greater number of follicles were aspirated using convex than to linear probe (12.4 versus 7.8, respectively, P<0.05). Mean number of oocytes and recovery rates were similar for convex (5.4 and 48.6%) and linear (4.6 and 59.3%) transducers. Limited space between the linear transducer and needle guide restricted access to some portions of the ovary, reducing the number of follicles aspirated using a linear transducer. The newly developed adaptor allowed greater stability, holding the ovaries firmly against the linear transducer. This diminished mobility permitted a similar number of oocytes to be recovered with both transducers. In conclusion, this new adaptor provided a low cost alternative for routine follicle aspiration and oocyte recovery in cattle.     

Evaluation of fluids from cystic follicles for in vitro maturation and fertilization of bovine oocytes

Follicular cysts are defined as cystic structures derived from unovulated follicles. The formation of the cysts appears to be related to failure of the oocyte to resume meiosis. The aim of this study was to evaluate in the bovine: 1) the ability of the fluid from cystic follicles to promote in vitro oocyte maturation and fertilization, 2) the predictive value of the morphology of oocytes derived from cystic follicles on the ability of the follicular fluid to promote in vitro maturation/fertilization as well as the oocytes to undergo maturation and fertilization. In Experiment 1, the ability of fluid from cystic (and normal) follicles from live and slaughtered cows (to promote) in vitro maturation and fertilization of bovine cumulus-oocyte-complexes (COC's) was assessed by cumulus expansion, sperm penetration, male pronucleus formation and polyspermy rates. Concentrations of progesterone (P4) and estradiol-17 beta (E2) were measured in the fluid from cystic follicles collected from live and slaughtered cows. In Experiment 2, we investigated the relationship of the morphology of COC's from cystic follicles, and the effect of the follicular fluids on oocyte maturation as well as P4 and E2 concentrations. In Experiment 1, although sperm penetration and male pronucleus formation were inhibited significantly by fluid from some cystic follicles collected from live and slaughtered cows, there were no significant differences in sperm penetration, male pronucleus formation and polyspermy rates between fluid from cystic follicles collected from live cows, from slaughtered cows and from control groups, regardless of the P4/E2 ratio. In Experiment 2, the morphology of cumulus-oocyte complexes from cystic follicles varied and the pronucleus formation of oocytes after in vitro fertilization was abnormal. On the other hand, the male pronucleus formation rates were not significantly different between the cystic follicular fluids and control, regardless, of the P4/E2 ratio. The results of this study suggest that many of the bovine follicular fluids from cystic follicles possess the ability to induce cumulus expansion, nuclear maturation and male pronucleus formation following in vitro maturation and fertilization of bovine oocytes. The morphology of the cumulus-oocytes complexes from cystic follicles seems not to relate to the ability of the cystic follicular fluids to induce oocyte maturation, and oocytes from cystic follicles possess the ability to form male pronucleus even though most were abnormal after in vitro fertilization.     

Thoracic fine needle aspiration biopsy versus fine needle cutting biopsy. A comparative study of 40 patients

The fine needle aspiration (FNA) biopsy findings were compared with the results of fine needle cutting (FNC) biopsy in 40 patients. The lesions (38 pulmonary nodules, 1 mediastinal mass and one lytic rib lesion) were biopsied with 22-gauge Greene and 21-gauge E-Z-EM needles through a 19-gauge needle guide. The FNA biopsy findings were based on smears and cell blocks of material obtained with the Greene needles while the FNC biopsy findings were based on tissue cores obtained by the E-Z-EM needles. In 83% of the cases, both techniques yielded specimens with similar cellularity; in seven cases, the FNA samples were more cellular. Malignancy was diagnosed in 80% of the patients: by both techniques in 26 patients, by FNA biopsy only in 5 patients and by FNC biopsy only in 1 patient. The sensitivity of FNA biopsy was higher than that of FNC biopsy (96.8% vs. 84.3%). The specificity and predictive value of positive results were 100% for both techniques. The predictive value of negative results was higher for FNA biopsy (88.8% vs. 54.5%). The majority of FNC biopsy tissue cores consisted mostly of clotted blood, lung tissue and/or fibrous tissue and did not facilitate or improve the diagnosis. Those data suggest that the contribution of FNC biopsy to the diagnosis of thoracic neoplasms is very limited and that the performance of FNC biopsy with an E-Z-EM needle in addition to or instead of FNA biopsy is not justified.     

The relationship of follicle atresia to follicle size, oocyte recovery rate on aspiration, and oocyte morphology in the mare

Oocytes were collected by aspiration of follicles from horse ovaries obtained at surgery or post-mortem. The oocytes were classified according to morphology of the ooplasm and cumulus. The size of the corresponding follicles was measured, and sections of the follicles were fixed and examined histologically to determine the stage of viability or atresia. In Part 1, 11 pairs of ovaries were examined and all follicles were sectioned; in Part 2, 9 pairs of ovaries were examined and only those follicles from which oocytes were recovered were sectioned. The number of follicles examined per pair of ovaries in Part 1 (average +/- SD) was 12.9 +/- 4.1. The proportion of follicles that were viable increased with increasing follicular size (P < 0.01); the percentage of viable follicles was 21, 42 and 83% for follicles < 10 mm, 10 to 19 mm, and >/= 20 mm in diameter, respectively. The overall oocyte recovery rate on aspiration of follicles was 46%. There was no significant difference in the oocyte recovery rate between viable and atretic follicles. A significantly higher proportion of oocytes recovered from viable follicles had granular ooplasm (64 vs 39%; (P < 0.05); whereas significantly more oocytes from atretic follicles had a misshapen or dense ooplasm (23 vs 6%; P < 0.05), or an expanded or pyknotic cumulus (24 vs 6%; P < 0.05). The most common cumulus morphology (63% of oocytes from viable follicles and 48% of oocytes from atretic follicles) was presence of only the corona radiata. Only 11% of oocytes from viable follicles and 9% of oocytes from atretic follicles had a complete cumulus present.     

Influence of the collection technique of prepubertal goat oocytes on in vitro maturation and fertilization

Prepubertal goat ovaries obtained from a slaughterhouse were used to study the influence of the oocyte collection technique (dissection, aspiration and slicing) on the number of oocytes recovered and their capacity for maturation and fertilization in vitro. The oocytes were recovered using 3 techniques, were selected for culture and were classified according to the number of cumulus cell layers. The numbers of oocytes selected per ovary were 1.71, 1.27 and 6.05 for dissection, aspiration and slicing, respectively. The percentages of maturation obtained for slicing (56.9%) were lower than those obtained for dissection and aspiration (69.3 and 72.0%, respectively). The proportion of oocytes with the most cumulus cell layers (complete cumulus) was greatest for oocytes recovered by dissection, but this had no influence on their capacity for nuclear maturation. The total percentage of fertilization was similar for oocytes obtained by dissection and by slicing, but the latter yielded a lower percentage of normal fertilization (29.1 vs 18.2%). Of the oocytes obtained by slicing, no difference was observed in the fertilization rate between oocytes with a partial cumulus and a complete cumulus. The decrease in maturation time from 27 to 25.5 and 24 h did not improve the results for fertilization but caused a decrease in the percentage of nuclear maturation. In conclusion, the recovery of oocytes using the slicing technique yielded more oocytes per ovary than dissection or aspiration, although the in vitro fertilization capacity of oocytes obtained by the slicing method was lower than for oocytes obtained by dissection.     

Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares

Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory follicles of estrous mares 32 to 36 h after administration of hCG; 2) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and matured in vitro for 36 to 38 h; 3) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and transferred into a recipient's oviduct <1 h after collection; and 4) im mature oocytes collected from slaughterhouse ovaries containing a corpus luteum and matured in vitro for 36 to 38 hours. Embryo development rates were higher (P < 0.001) for oocytes matured in vivo (82%) than for oocytes matured in vitro (9%) or within the oviduct (0%). However, neither the method of maturation nor the source of oocytes affected (P > 0.1) embryo development rates after the transfer of immature oocytes.     

In vitro production of bovine embryos: developmental competence is acquired before maturation

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.     

Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro

Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus cells.