In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis).

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.     

Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers

In the present study, comparison of 2 different culture media (Ham's F-12 and M-199) for supporting in vitro maturation of goat oocytes, and their subsequent embryonic development was evaluated in the presence or absence of sera (estrous goat serum, EGS and fetal calf serum, FCS) and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol, 1 ug/ml). Neither medium (Ham's F-12 or M-199) when supplemented with EGS and hormones showed any notable changes in the maturation rate nor in cleavage and blastocyst development. The mean cell number for blastocysts was also significantly low (P < 0.05). However, Ham's F-12 medium supplemented with FCS and hormones showed a considerable increase in the maturation rate, but subsequent embryonic development was not appreciably increased. However, maturation, cleavage and blastocyst development rates of oocytes matured in M-199 medium in combination with 10% FCS and hormones were significantly higher (P < 0.05). Mean cell number per blastocyst was also significantly increased in this latter treatment compared with that of the other groups (P < 0.05). The results thus indicated that both the culture medium and serum have a marked effect on maturation and subsequent embryonic development. Further, the results also showed that the combination of M-199 with FSH, LH and E2 supplemented with 10% FCS was the most efficacious medium for in vitro maturation and subsequent embryonic development of the media, sera and hormone combinations studied.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Methods for collecting and maturing equine oocytes in vitro

The objective of our study was to develop an effective method for collecting and maturing equine oocytes. In Experiments 1 and 2, oocytes were collected from excised ovaries obtained via colpotomy. In Experiment 3, oocytes were collected from ovaries obtained after slaughter. Follicles were aspirated and flushed with various treatments to recover the oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0.5 ml of modified Dulbecco's PBS with 0, 100 or 500 lU/ml hyaluronidase. There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Ham's F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 17β, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P<0.05) at 36 h for oocytes cultured in TCM-199 (79%) than for those in Ham's F-10 (21%). There was no difference (P>0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PBS or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultured in TCM-199 for 24, 30 or 36 h. There was an increase (P<0.05) in oocyte recovery when follicles were flushed with PBS, with or without hyaluronidase. There was also a difference (P<0.05) in the percentage of maturation of oocytes between 30 and 24 h (86 vs 48%), but no further increase was seen by 36 h (84%). In Experiment 3, follicles were aspirated with PBS 5 to 6, 6 to 7 or 7 to 8 h after slaughter. The oocytes were cultured for 30 h in TCM-199 either with or without 100 IU/ml eCG. There was no effect of eCG or time from slaughter on oocyte maturation or cumulus expansion (P>0.05).     

The effect of the composition of the culture media on bovine oocyte maturation and embryo development in vitro

We studied the capacity of the cattle oocyte for the resumption of meiosis and achievement of metaphase II in various protein-free culture media (DMEM, TCM-199, Ham's F-10, and Ham's F-12) and the pattern of influence of the estrous serum on in vitro development of fertilized cattle oocytes, with special reference to the time of its addition to the synthetic oviduct fluid containing BSA. In the first experimental series, it was shown that the highest number of oocytes (76.1%) resumed meiosis in DMEM medium. Meiosis was not resumed in Ham's F-12. Intermediate results were obtained for TCM-199 (55.1%), which is commonly used for maturation of cattle oocytes in vitro, and for Ham's F-10 (51.7%). The oocytes reached metaphase II in DMEM at a higher rate (45.3%) than in TCM-199 or Ham's F-10 (29.4 and 8.6%, respectively). In the second experimental series, the estrous serum was added to the culture medium within 20 h (control) or 42 h (experiment) after the beginning of fertilization. The estrous serum did not inhibit the first cleavage division (the percentage of cleaving embryos did not differ reliably: 32.7 and 37.9%, respectively). However, a later serum addition to the culture medium (within 42 h after the beginning of fertilization) reliably increased the percentage of embryos that reached the blastocyst stage (6.5% in the control and 17.8% in the experiment) and the hatched blastocyst stage (2 and 9.2%, respectively).     

Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos

The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10microg/ml FSH and group (3) in which IVM medium was supplemented with 10IUml(-1) eCG. In all experiments, oocytes were kept at 38.5 degrees C under 5% CO(2) for IVM, IVF, IVC and examined for cleavage and embryo development rates on days 3 and 8, respectively. Good and fair quality oocytes produced a higher cleavage rate (P<0.01) than poor quality oocytes. Morula production rate was also higher (P<0.01) for good as compared with fair quality oocytes. Embryo development with poor quality oocytes was arrested at the two to sixteen cell stage. In experiment 2, the cleavage rate was higher (P<0.05) in CR1aa than CR2aa, and higher (P<0.01) than TCM-199, MEM and RPMI-1640. The numbers of morulae and blastocysts were higher (P<0.01) for oocytes cultured in CR1aa and CR2aa media than TCM-199 or MEM. In experiment 3, the addition of FSH or eCG to the maturation medium increased (P<0.01) cleavage and developmental rates of buffalo embryo compared with control medium. In conclusion, the IVM of good quality buffalo oocytes in CR1aa or CR2aa medium and the addition of FSH or eCG in maturation medium produced higher cleavage and developmental rates of IVF buffalo embryos.     

In vitro maturation and fertilization of goat oocytes

Goat oocytes were isolated from 3-5 mm diam. follicles. The oocytes with compact cumulus mass were matured and fertilized in vitro. Three different media, viz. modified Krebs-Ringer bicarbonate, Dulbecco's and Ham's F-12 with three different additives (bovine serum albumin, BSA; follicle stimulating hormone, FSH and fetal calf serum, FCS) were tested. The three basal media gave almost similar results with Ham's F-12 being slightly better. Addition of BSA (10 mg/ml) increased the rates of maturation and penetration. FSH + BSA (2.5 micrograms/ml + 10 mg/ml) further enhanced the rates while FCS (10%) proved to be even more effective. In modified Krebs-Ringer bicarbonate and Dulbecco media with additives FCS + BSA, around 60% oocytes matured to metaphase II of which 53% were penetrated by capacitated goat spermatozoa while in F-12 medium 70% reached metaphase II and 63% were penetrated. Ham's F-12 medium with additives FCS + BSA was slightly better for maturation and penetration of goat oocytes in comparison to two other media tested.     

Developmental potential of rabbit oocyte matured in vitro: the possible contribution of prolactin

The present study was undertaken to define hormonal conditions for in vitro maturation that support subsequent fertilization and embryonic development. Follicular oocytes were recovered from nonstimulated rabbit ovaries and cultured for 12 h in Brackett's medium supplemented with or without hormones. Matured oocytes were inseminated in vitro and transferred 12 h later to Ham's F-10 medium supplemented with 20% fetal calf serum. The initial cleavage frequency of matured oocytes in Brackett's medium was comparable to the frequency of development for in vitro-matured oocytes under various hormonal conditions. However, the addition of estradiol (E2, 1 microgram/ml) to incubation medium containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased significantly (p less than 0.001) the percentage of embryos achieving morula or blastocyst formation (16/98, 16.3%), as compared to the mature oocytes in medium containing LH, LH plus FSH, or no hormone. The addition of prolactin (PRL) to the maturation medium increased the percentage of development to organized embryos in a dose-dependent manner. In vitro-matured oocytes in medium containing LH, FSH, and PRL exhibited a significantly (p less than 0.001) lower incidence of developmental competence (5/95, 5.3%) than oocytes matured in the presence of E2 in conjunction with pituitary hormones (43/89, 48.3%). These results demonstrate that hormonal composition in the environment of the oocyte is critical for acquisition of developmental capacity. PRL as well as E2 appears to be an important constituent in the process of oocyte maturation, promoting preimplantation embryonic development.     

Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes

The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.     

Effect of including growth factors and antioxidants in maturation medium used for in vitro culture of buffalo oocytes recovered in vivo

This study examined the effect of including one of two growth factors (100 ng/ml IGF-1 or 20 ng/ml EGF) in combination with one of two antioxidants (50 microM cysteamine or 50 microM beta-mercaptoethanol) in maturation, fertilization and subsequent development of buffalo oocytes. The oocytes were recovered by in vivo ovum pick-up technique from six Murrah buffalo heifers twice a week over a period of 16 weeks. Immediately after ovum pick-up oocytes recovered from six donors were allocated randomly to five different maturation treatments. The control treatment was the basic maturation medium (MM; TCM-199 supplemented with 10% FBS, 10 IU/ml LH, 0.5 microg/ml FSH, 1 microg/ml estradiol-17beta and 50 microg/ml gentamicin). The other four treatments consisted of the control maturation medium (MM) plus one combination of a growth factor and an antioxidant viz. IGF-1+cysteamine; IGF-1+beta-ME; EGF+cysteamine or EGF+beta-ME. The total number of oocytes assigned to each maturation treatment ranged from 31 to 66. After maturation in different maturation medium, media used for in vitro fertilization and subsequent development of embryo was same for all groups. Data were analysed using Chi-square test. The maturation rate observed for the growth factor plus antioxidant treatments was similar to that for the control (90.4%). The highest cleavage rate recorded in the IGF-1+cysteamine treatment (71.9%) which was significantly higher (P<0.05) than the IGF-1+beta-ME (45.2%) and EGF+beta-ME (46.4%) treatments, but not significantly differ from the control (63.8%) and EGF+cysteamine treatment (60.7%). The proportion of cleaved oocytes those developed to blastocyst stage was significantly higher in the IGF-1+cysteamine treatment (52.2%; P<0.05) than in the control (23.3%), the EGF+cysteamine (13.5%) or the EGF+beta-ME (7.7%) treatments, but did not differ significantly from the IGF-1+beta-ME (28.6%) treatment. Following non-surgical transfer of 15 embryos to 14 synchronized recipients, four became pregnant and only one recipient sustained the pregnancy as long as 4.5 months when spontaneous abortion occurred. It was concluded that supplementing the maturation medium with IGF-1+cysteamine improved the production of buffalo embryos significantly in vitro culture.     

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.     

Effects of gonadotropins on bovine oocytes matured in TCM-199

The effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) during in vitro maturation of bovine oocytes in TCM-199 without serum were evaluated. Bovine oocytes with compact cumulus cells were collected from slaughterhouse-derived ovaries and cultured in Hepes-buffered TCM-199 supplemented with 5 mg/mL BSA, 1 microg/mL estradiol-17beta, FSH (0, 0.015, 0.05, 0.15, 1.5 or 15 ng/mL; Experiment 1), LH (0, 0.14, 1, 7 or 49 microg/mL; Experiment 2) and combinations of 1 or 10 ng/mL FSH and 1 or 10 microg/mL LH (Experiment 3) at 39 degrees C in 5% CO2 in air. After 22 h of maturation, cumulus expansion was estimated by scoring from 0 (no expansion) to 4 (full expansion of cumulus mass). In vitro fertilization was done with Percoll (45/90%) separated bull sperm at 1 x 10(6) sperm/mL in fert-TALP with 5 U/mL heparin. At 18 to 20 h post-insemination, presumptive zygotes were transferred to a chemically defined medium (CDM-1) supplemented with 0.5% BSA and nonessential amino acids for 72 h and then moved to CDM-2, additionally supplemented with essential amino acids. Zygotes were cultured at 39 degrees C in 5% CO2, 5% O2 and 90% N2 for 8 days. During Experiments 1 and 2, cumulus expansion increased in proportion to concentrations of FSH and LH. Cleavage rates and development to blastocysts were not significantly different among FSH and LH treatments. In Experiment 3, cumulus expansion of bovine oocytes was maximal when 1 ng/mL FSH and 1 microg/mL LH were added to IVM medium, but cumulus expansion again was not related to developmental ability, although cleavage rates were improved slightly (P<0.05) by the combination of LH and FSH. Blastocyst quality, estimated by the size of inner cell mass, was not different between combinations of FSH and LH, and the numbers of nuclei were not different. Although expansion of cumulus cells surrounding bovine oocytes was altered in response to FSH and/or LH in semi-defined medium, cumulus expansion was not related to rates of cleavage or subsequent embryonic development in vitro. The effects of LH on cumulus expansion can be explained by as little as 1 part per 10, 000 contamination with FSH.     

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes

The influence of estrous cow serum (ECS) or fetal calf serum (FCS) and their interaction with gonadotropins on in vitro bovine maturation and fertilization was evaluated. The addition of ECS or FCS to the medium significantly increased the percentage of oocyte maturation over that of Ham's F-10 medium alone (P<0.05). The addition of follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) or of FSH plus hCG to the medium provided no advantage in maturation over serum alone. However, FSH plus HCG added to the maturation medium significantly improved the frequency of pronucleus formation in both groups compared with a medium to which no gonadotropins were added (P<0.05). Both ECS and FCS (as medium supplements) promoted oocyte maturation. Although the addition of a combination of FSH plus hCG to the medium did not increase the rate of oocyte maturation, it did contribute to the high incidence of subsequent pronucleus formation.     

Ovarian responses and oocyte recovery in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH or PMSG treatment

Stimulation of follicular growth was examined using two different gonadotropin treatments in 10 prepubertal swamp buffalo calves (8 to 12 mo old). Each calf received an ear implant consisting of 3 mg norgestromet and 5 mg estradiol valerate during hormonal treatment. Five calves were additionally administered FSH (24 mg, im) and, 2 mo later, PMSG (3,000 IU). The remaining 5 calves were first treated with PMSG followed by FSH. Ovarian responses to treatments were examined by laparotomy, 72 h after ear implant removal, and by the number of follicles (diameter > or = 0.8 cm) and corpora hemorrhagica present. Ovaries had more significant response to FSH than PMSG treatment (13.9+/-8.6 vs 5.9+/-3.3 follicles; P<0.01). Although the recovery rate tended to be lower for FSH treated (64%) than PMSG-treated (82%) animals, more oocytes/animal were harvested in the PMSG treatment (8.3+/-5.0 vs 4.6+/-3.2, respectively). The immature oocytes (n = 38) were cultured for 24 to 25 h in maturation medium (TCM-199 NaHCO3+10% fetal calf serum [FCS] in 5%CO2 in air at 39 degrees C). Oocyte maturation was assessed after fixation and staining with aceto orcein. The in vitro maturation rate was 52.6% (20/38). This study shows the possibility of harvesting oocytes from prepubertal swamp buffalo calves and maturing the oocyte in vitro.     

Luteinizing hormone-enhanced in vitro maturation of bovine oocytes with and without protein supplementation

Luteinizing hormone was shown to enhance maturation of immature oocytes obtained from slaughtered cattle as reflected by elevated proportions of oocytes that fertilized and reached blastocyst stages in vitro after in vitro fertilization (IVF). Higher proportions of ova were fertilized in vitro after in vitro maturation (IVM) in modified TCM-199 (TCM-199 + BSA + LH [USDA-bLH-B-5, 100 micrograms/ml]) than in TCM-199 alone (p less than 0.01). Further improvement in IVF (p less than 0.005) followed IVM when 20% proestrous (Day 20) bovine serum replaced the BSA, but similar proportions of inseminated ova (22.2% and 22.6%) developed into blastocysts. The positive LH effect was verified in defined conditions for IVM. Exposure of oocytes to the purified LH preparation (without any other added protein or biological substances) during IVM improved IVF (39.7% in TCM-199 vs. 73.5% in TCM-199 + LH; p less than 0.001) and blastocyst development (7.9% vs. 28.2%; p less than 0.005), respectively. Efforts to better define effective concentrations of LH revealed no difference in viability after IVM with 50 micrograms LH/ml vs. 100 micrograms LH/ml (27.0% vs. 28.3%, respectively); 10 micrograms LH/ml did not enhance viability when compared to TCM-199 alone (10.8% vs. 9.9%). Results demonstrate potential utility of this approach for investigation of factors influencing mammalian development by specific effects initiated during the interval of oocyte maturation.     

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.     

Effect of sperm exposure time on in vitro fertilization and embryo development of bovine oocytes matured in vitro

This study was designed to investigate the effect of sperm exposure time on the fertilization rate and subsequent developmental capacity of bovine oocytes matured in vitro. Cumulus oocyte complexes (COCs) obtained from 2 to 6 mm follicles were matured for 24 h in TCM-199 supplemented with fetal bovine serum (FBS) and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed by incubating 15 to 20 matured oocytes with 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium for either 4, 8, 12, 16, 20, 24 or 28 h. Following sperm exposure for different periods of times, the presumptive zygotes were co-cultured with Buffalo Rat Liver cells (BRLC) monolayers in CZB medium without glucose, a simple semi-defined medium developed for mouse embryo culture, for 3 d post-insemination and then in M199/FBS (TCM-199-HEPES supplemented with 20% heat-treated FBS and 1 mM sodium pyruvate) for 5 d. The fertilization rates differed significantly among the 7 treatment groups, with higher frequencies obtained by co-incubation of gametes for 20, 24 or 28 h (67 to 76%) than for 4, 8 and 12 h (26 to 54.5%), with 16 h (57%) being intermediate. However, the length of sperm exposure time did not significantly affect subsequent embryo development, although an increasing trend was noted from 4 h to 20 h. The number of fertilized oocytes at 3 d post-insemination cleaving to 2- to 4-cell vs 8-cell stage was not different among treatment groups. Development of 8-cell embryos to morulae and blastocysts did not differ among the treatment groups. These data suggest that the optimum duration of sperm-oocyte incubation is 24 h, and periods shorter than 16 h may result in a reduced fertilization rate.