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.     

Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer (Cervus elaphus) oocytes

Immature red deer (Cervus elaphus ) oocytes (n = 1208) were collected from 1 to 4 - mm diameter follicles on ovaries and then cultured for 16, 20, 24 or 28 h (Groups I to IV) in TCM 199 supplemented with 10% FCS, 1 x 10(6) granulosa cells/ml and 1 mug/ml estradiol at 39 degrees C under 5% CO(2) in air. Gonadotropins (10 mug/ml, FSH and LH) were added to the culture medium at the start of culture (0 h) or after 6 h. Approximately one-third of the oocytes were examined for maturation, and the remainder were fertilized in vitro with frozen-thawed semen collected from a stag by electroejaculation. In vitro fertilized oocytes (n = 309) from four of the maturation treatment (Groups II and III in both gonadotropin treatments) were cultured for 7 d and examined for cleavage. Oocytes cultured for 16 h (Group I) had lower (P < 0.001) maturation rates (4.7%) than those in the longer culture durations (Groups II to IV: 68.9%). Culture for 20 (Group II) and 24 h (Group III) resulted in higher (P <0.001) fertilization rates than culture for 16 (Group I) and 28 h (Group IV) (18.3, 20.5, 7.1, 7.8%, respectively). The time of gonadotropin addition did not affect maturation or fertilization rates, but its addition at 6 h increased (P < 0.05) the percentage of oocytes cleaving (5.7 vs 12.5%). Oocytes cultured for 20 h (Group II) and with the delayed addition of gonadotropins cleaved most readily (18.2%). No embryos developed beyond eight-cell stage.     

Term development of caprine embryos derived from immature oocytes in vitro

Ovaries were surgically removed from female goats (Toggenburg, Nubian and Saanen breeds). Oocytes were collected by follicular aspiration or after ovaries were minced, then matured in mTCM-199 with 100 mug LH + 0.5 mug FSH + 1.0 mug estradiol 17-beta/ml for 27 h prior to in vitro fertilization (17). Although more oocytes were made available by mincing than by aspiration, higher proportions of aspirated oocytes were fertilized and developed to morulae. Proportions that fertilized and reached morulae were 82 102 (80.4%) and 50 102 (49.0%) versus 77 126 (61.1%) and 27 126 (21.4%) for oocytes obtained by aspiration and after ovarian mincing, respectively (P<0.05). Proportions of inseminated ova undergoing cleavage and continuing development to the morula stage differed significantly (P<0.05) among 5 co-culture treatment groups, with higher proportions of cleavage (23 27 , 85.2%) and morulae (14 27 , 51.9%) obtained by co-culture on caprine cumulus cells (cCC). Some oocytes reached the blastocyst stage (4 54 , 7.4%) following oocyte collection by aspiration and culture on caprine oviduct epithelial cells (cOEC). After 4- and 8-cell stage embryos obtained by aspiration and culture on cCC were transferred pregnancy resulted. Twin male kids (developed from different embryos) were born on August 6, 1993, and have developed into normal bucks. Conditions reported here provided an adequate environment for support of oocyte maturation, fertilization and early embryonic development in vitro (IVMFC) with normal development after embryo transfer.     

Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.     

Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes

Experiments were conducted to evaluate the effects of cooling porcine ovaries to low temperature (4 degrees C, 15 degrees C, 20 degrees C, 25 degrees C or 30 degrees C) for 1 h on the meiotic competence of their oocytes. Moreover, it was determined whether or not the exposure of in vitro matured oocytes to ambient temperature (20 degrees C, 25 degrees C or 30 degrees C) for 1 h affects the fertilization and developmental competence of the oocytes. There was no difference between the proportions of oocytes that underwent maturation to metaphase II when isolated from control ovaries held at 35 degrees C and ovaries exposed to 30 degrees C. However, the percentages of oocytes from ovaries exposed to 25 degrees C or less were significantly lower than those of oocytes from ovaries exposed to 30 degrees C and control ovaries. The proportions of total and normal fertilization of oocytes that had been exposed to 20 degrees C before in vitro fertilization (IVF) were significantly lower than those of control oocytes maintained at 38.5 degrees C. However, cooling in vitro matured oocytes had no effects on their cleavage and development to blastocysts after IVF. These data suggest that exposing porcine ovaries to a low temperature of 25 degrees C or less before aspiration of oocytes may adversely affect their subsequent in vitro maturation. It may be necessary to maintain the oocytes at a temperature of more than 25 degrees C during manipulation of oocytes for retaining the fertilizability of the oocytes.     

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.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

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).     

Cryopreservation of equine oocytes by 2-step freezing

Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.     

A comparison of three methods of recovery of goat oocytes for in vitro maturation and feritilization

Experiments were conducted to determine an efficient method for recovering a large number of usable oocytes and its effect on subsequent in vitro maturation, fertilization and development. Follicular oocytes were recovered from goat ovaries using 3 different methods: aspiration, puncturing and slicing. The average total number of oocytes recovered per ovary was significantly higher by the aspiration method (2.7+/-0.15) than by puncturing (2.2+/-0.13) or by slicing (2.4+/-0.12). However, significantly more good-quality usable oocytes enclosed with compact cumulus cells were obtained by slicing (0.9+/-0.06) than by aspiration (0.5+/-0.07) or by puncturing (0.5+0.06). Time required for processing each ovary by the slicing method was comparatively less (0.90 min) than that required for puncturing (1.83 min) or for aspiration (1.78 min). Usable oocytes recovered by all three methods were matured, fertilized and developed to the blastocyst stage in vitro. There were no significant differences in the subsequent percentages of oocytes maturing, being fertilized and developing in vitro among the 3 methods of recovering oocytes. In conclusion, the recovery of goat oocytes by the slicing method is simple and efficient compared with the aspiration and puncturing methods.     

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.     

Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.     

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).     

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.     

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection

The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1) for 10 min; (d) 250 micromol inositol 1,4, 5-triphosphate l(-1) injection; and (e) no treatment (control). After 18-20 h further culture, the cumulus-oocyte complexes were assessed for activation by observing whether they had progressed through second anaphase-telophase and had formed a female pronucleus. The proportions of oocytes activated after each treatment were: 16/27 (59%) for ionomycin; 14/25 (56%) for ethanol; 22/28 (79%) for thimerosal; 15/27 (56%) for inositol 1,4,5-triphosphate; and 0/20 (0%) for the untreated controls. Thus, significantly more oocytes (P < 0.05) were activated by treatment with thimerosal than by the other four treatments. The proportions of oocytes that cleaved to the two-cell stage at 24-30 h after sperm injection in the groups treated with ionomycin, ethanol and thimerosal were 7/20 (35%), 5/19 (26%) and 11/23 (48%), respectively. No cleavage was observed in any of the control oocytes or those treated with inositol 1,4, 5-triphosphate. Furthermore, evidence of normal fertilization was observed in 2/7 (29%), 2/5 (40%) and 7/11 (64%) of the oocytes treated with ionomycin, ethanol and thimerosal, respectively. These results demonstrated that: (a) it is possible to activate equine oocytes with the chemical stimulants, ionomycin, ethanol, thimerosal and inositol 1,4,5-triphosphate; (b) thimerosal is more effective than the other three reagents in facilitating both meiotic activation and normal fertilization of equine oocytes; and (c) chemical activation may also stimulate parthenogenetic cleavage of oocytes without concurrent changes in the head of the spermatozoon.     

Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes

The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.     

Cytogenetic evaluation of in vitro matured bovine oocytes collected from ovaries of individual donors

Oocytes were collected after slaughter by aspiration from pairs of ovaries of individual donors. A total of 656 oocytes was selected for IVM from 74 pairs of ovaries (8.9 oocytes per pair, ranging between 1 and 25). The oocytes were matured in droplets of maturation medium (TCM-199 medium supplemented with 20% estrous cow serum (ECS), 50 microg/ml gentamycin, 10 microg/ml FSH, 1 microg/ml estradiol-17beta). Cytogenetic analysis of 348 oocytes showed 79 at the first metaphase (MI; 22.7%, 79 348 ), 11 at the first telophase (TI; 3.2%, 11/348 ), and 258 at the second metaphase (MII; 74.1% 258/348 ). Significant differences (P < 0.01) were shown among the donors regarding the number of oocytes selected for IVM and the number of oocytes matured for IVF.     

小鼠卵巢冷冻移植后卵泡发育和卵母细胞成熟的研究

本研究探讨了冷冻保存的1日龄小鼠卵巢异体异位移植后,其原始卵泡重新启动生长发育的能力。一日龄B6C2F．小鼠卵巢分离冷冻后置液氮中保存,保存1周。6个月后解冻,并将卵巢移植到8-12周龄B6C2F．受体鼠。肾脏包膜下,移植至少14d。每侧肾囊移植2枚卵巢的40只受体鼠中卵巢的回收率为45．00％（72／160）,而每侧。肾囊移植l枚卵巢的20只受体鼠的回收率为82．50％（33／40）。移植卵巢上卵泡的发育基本与体外自然生长鼠的卵巢卵泡发育情况一致。对卵巢移植19d的受体鼠用孕马血清促性腺激素（pregnant mare serum gonadotrophin,PMSG）处理后,从移植卵巢上发育成熟卵泡中获得的卵母细胞在MEM0c培养基中培养16-17h,有40．90％的卵母细胞发生生发泡破裂（germinal vesicle breakdown,GVBD）,其中89．02％的卵母细胞发育到第二次减数分裂中期（metaphaseⅡ,MⅡ）。将剩余的卵母细胞继续培养到20～21h,又有50．83％的卵母细胞发生生发泡破裂,但其中只有21．40％的卵母细胞能够发育到MII期。以上结果说明,小鼠早期卵巢经过冷冻．解冻并异体异位移植后,其原始卵泡能够重新启动生长发育,发育后的卵泡卵母细胞能够在体外培养成熟。这些结果意味着原始卵泡或卵巢冷冻一移植技术有可能充分利用雌性生殖细胞用于濒危动物保种、建立动物基因库和人类辅助生殖等。