Estrous Cow Serum Research Articles

Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro.

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.

Fecundação in vitro de ovócitos bovinos com sêmen submetido a diferentes diluidores

O objetivo deste trabalho foi avaliar a influência do diluidor do sêmen no desenvolvimento in vitro de ovócitos bovinos após a maturação e fecundação in vitro. Ejaculado de um reprodutor foi fracionado e submetido a três diluidores: Lactose/gema de ovo (LG), Citrato/gema de ovo (CG) e Tris/gema de ovo (TG). Amostras deste material foram envasadas, congeladas e estocadas em N² e, posteriormente, descongeladas; a fração móvel foi separada por gradiente descontínuo de Percoll. A concentração espermática foi ajustada para 10 x 10(6)/mL e a capacitação espermática, induzida com 10 µg/mL de heparina. Após 24 horas de cultura para maturação in vitro, os ovócitos, aspirados de folículos ovarianos, foram inseminados com sêmen diluído em meio TALP e, após 48 horas de cultura, os zigotos foram transferidos para gotas de meio TCM 199, com 5% de soro fetal bovino, 5% de soro de vaca em estro e suspensão de células epiteliais do oviduto bovino, cobertas com óleo de silicone, e mantidos em cultura por nove dias. Todas as culturas foram realizadas a 38,5ºC em atmosfera com 5% de CO2. Os dados foram analisados pelo teste do qui-quadrado e houve diferença com relação à taxa de clivagem (TC), sendo as médias de 66,0; 69,3; e 54,4% para LG, CG e TG, respectivamente. Não houve diferença entre tratamentos com relação às taxas de mórulas/blastocistos ou de eclosão. O diluidor do sêmen não teve efeito sobre o desenvolvimento in vitro de embriões bovinos, embora a TC tenha sido afetada.

In vitro production of embryos alters levels of insulin-like growth factor-II messenger ribonucleic acid in bovine fetuses 63 days after transfer.

The objective of this study was to determine the effect of embryo production systems on the expression of insulin-like growth factor (IGF)-II mRNA in fetal bovine tissues at Day 70 of gestation (63 days after transfer). Oocytes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. Zygotes were cultured in either tissue culture medium (TCM)-199 + 10% estrous cow serum (ECS; in vitro-produced with serum [IVPS]) or TCM-199 + 1% BSA (in vitro-produced with serum restriction [IVPSR]). At 72 h postinsemination, IVPSR embryos were transferred into fresh TCM-199 + 10% ECS whereas IVPS embryos had fresh medium replaced. All embryos were cultured for an additional 96 h. In vivo-produced embryos were harvested from superovulated Holstein cows (multiple ovulations [MO]). Grade 1 blastocysts from all groups were transferred singly into Angus heifers. At Day 70 of gestation, fetuses (n = 14, 13, and 11 for MO, IVPS, and IVPSR, respectively) were collected; liver and skeletal muscle samples were snap frozen, and whole-cell RNA (wcRNA) was extracted. Levels of IGF-II mRNA were determined by RNase protection assay and quantified relative to 18S rRNA (mean arbitrary units +/- SEM). WcRNA from adult and Day 90 fetal bovine liver were used as controls. Adult liver contained 9-fold less IGF-II mRNA than liver from Day 90 fetuses (P < 0.05). Fetal livers of males originating from IVPS and IVPSR groups possessed approximately 2-fold greater levels of mRNA for IGF-II than those from MO males (0.25 +/- 0.07, 0.33 +/- 0.04, and 0.14 +/- 0.03, respectively; P < 0.05). Levels of mRNA for IGF-II tended to be lower (P = 0.07) in skeletal muscle of fetuses originating from the IVPSR group (0.043 +/- 0.005) compared to MO controls (0.070 +/- 0.008). In conclusion, at Day 70 of gestation, fetuses originating from in vitro production systems possessed altered levels of IGF-II mRNA in both liver and skeletal muscle.

Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro

The present work describes a cytogenetic study of in vitro-matured bovine oocytes designed to analyze the incidence of diploid oocytes induced by concentration of serum in the culture medium, follicle size, culture temperature and incubation time. In Experiment 1, immature follicular oocytes from follicles of the same size were cultured for 24 h in TCM-199 supplemented with increasing concentrations 0, 10, 20 and 50% of estrous cow serum (ECS). In Experiment 2, immature oocytes harvested from follicles of different sizes were cultured for 24 h in TCM-199 supplemented with 20% ECS at 39°C in 5% CO 2. In Experiment 3, immature follicular oocytes were matured in TCM-199 supplemented with 20% ECS at 2 different temperatures (37°C or 39°C) in 5% CO 2. In Experiment 4, immature oocytes were matured over 4 different incubation times (24, 36 and 48 h) in TCM-199 supplemented with 20% ECS in 5% CO 2. The highest concentration (50%) of ECS supplement in the culture medium induced the highest incidence of diploid oocytes. This incidence of diploid oocytes matured in vitro was higher in oocytes from follicles with a diameter between 11 and 15 mm. Finally, lower culture temperature (37°C) and prolonged incubation time (48 h) also significantly (P<0.01) increased the percentage of diploid oocytes.

Comparison of the effect of two bovine in vitro culture systems, TCM199/FCS/BRL-co-culture and SOFaaBSA, on embryo production and pregnancy rate

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39°C under 5% CO2 in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69125), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (6569), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.

Cytogenetic analysis of bovine parthenotes after spontaneous activation in vitro

We conducted a cytogenetic study of bovine parthenotes derived from oocytes matured and cultured in vitro. In vitro maturation was carried out by culturing follicular oocytes for 24 h in TCM199 supplemented with estrous cow serum (ECS) and hormones at 39 °C in 5% CO 2. Matured oocytes were incubated for 20 h in sperm TALP without the addition of spermatozoa, after which they were cultured in maturation droplets for 48 to 72 h. Spontaneous activation occurred in 9.5% of the matured oocytes. Cytogenetic analysis of 24 parthenotes revealed that 62.5% exhibited a normal, diploid chromosome complement. The remaining 37.5% had various ploidy anomalies: haploidy (25%), triploidy (4.2%) and tetraploidy (8.3%). Parthenotes exhibited different developmental stages. The number of blastomeres ranged from 2 to 8 within a parthenote. Only 1 parthenote was comprised 9 to 16 cells. The results showed that spontaneous parthenogenetic activation which occurs in an IVM/IVF system may interfere with embryo production efficiency.

Modulatory actions of activin-A and follistatin on the developmental competence of in vitro-matured bovine oocytes.

The presence of activin receptors on oocytes and granulosa cells suggests that activin and its binding protein, follistatin, may regulate oocyte maturation. The aim of the present study was to investigate whether activin-A and follistatin can influence the in vitro maturation of bovine oocytes as assessed by their competence to form blastocysts after in vitro fertilization. Bovine cumulus oocyte complexes (COCs) were cultured for 22-24 h at 38.5 degrees C in tissue culture medium-199 supplemented with 10% estrous cow serum, eCG (2.5 IU/ml), and either no treatment (control), activin-A (0.1 or 0.5 microg/ml), follistatin (0.1, 1, or 10 microg/ml), or activin-A (0.5 microg/ml) in combination with follistatin (0.5 or 5 microg/ml). In separate experiments, the same treatments were also tested on cumulus-free oocytes, which had a much reduced developmental capacity when compared to COCs. Neither activin-A nor follistatin affected the postfertilization cleavage rate of either COCs (approximately 60%) or cumulus-free oocytes (approximately 40%). Activin increased (p < 0.05) the proportion of cleaved oocytes that reached the blastocyst stage when added to both COCs (38% increase) and cumulus-free oocytes (160% increase), with the magnitude of response much greater with the latter. Follistatin had a dose-dependent inhibitory effect on blastocyst yield from COCs (67% reduction, p < 0.05) and opposed the stimulatory effect of activin (p < 0.05). With cumulus-free oocytes, however, follistatin did not further decrease the low developmental potential of oocytes. A positive correlation (r = 0.57, p < 0.001) was found between endogenous levels of activin-A produced by COCs and their postcleavage development to the blastocyst stage. No such correlation was found between endogenous follistatin level and postcleavage development (r = 0.10, p = 0.46). Endogenous levels of activin-A and follistatin secreted by cumulus-free oocytes were undetectable. These in vitro observations support the hypothesis that activin-A and follistatin, both secretory products of cumulus cells, contribute to the regulation of oocyte maturation in vivo.

Effect of exposure to cryoprotectant solutions on cleavage and subsequent development of immature bovine oocyte in vitro

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39°C under 5% CO2 in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69125), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (6569), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.

Avaliação das condições de maturação oocitária e do efeito do reprodutor na produção in vitro de embriões bovinos

The conditions of in vitro maturation of bovine oocytes (Exp. I) and bull effect (Exp. II) on cleavage (CR) and the in vitro morulae and blastocysts (MO/BL) rates were evaluated. Imatured oocytes were matured in vitro under different conditions: B-199 medium with 5% estrous cow serum (ECS), 5% fetal calf serum (FCS) and granulosa cells (T1/Exp. I) or B-199 medium with 10% FCS, 14 IU/ml PMSG and 7 IU/ml hCG (T2/Exp I). After 24 h culture, the mature oocytes were inseminated with frozen-thawed semen from two bulls (R1 and R2/Exp. II). In the Exp. I, the T1 and T2 oocytes were inseminated with semen from R1/Exp. II and in the exp. II the semen from both bulls inseminated T1/Exp. I oocytes. After 48 h, the 2-4 cells embryos were transferred into embryo culture droplets and were further cultured for 9 days. The CR of T2/Exp I was higher (p<0.01) than T1/Exp. I. There was a significant difference (p<0.05) between bulls on CR and Mo/Bl production. The use of hormones in a maturation medium has no effect on embryo production, however the procedure seems to be simpler than with granulosa cells. The use of semen from different bulls for in vitro fertilization has affected the subsequent in vitro production of embryos.

Evaluation of mitomycin-treated vero cells as a co-culture system for IVM/IVF-derived bovine embryos

Support of the in vitro development of IVM/IVF-derived bovine embryos by Vero cells was evaluated by comparing the following treatment groups: 1) proliferating (Unt-Vero) vs nonproliferating (Mit-Vero) cells; 2) supplementation of medium with estrous cow serum (ECS) vs bovine serum albumin (BSA); 3) Mit-Vero cells vs bovine oviduct epithelial cells (BOECs); and 4) addition of leukemia inhibitory factor (LIF) to Mit-Vero cell co-cultures at Day 1 vs Day 4. Mit-Vero cells stimulated higher rates of blastocysts (Day 7, 40 vs 27%) and hatched blastocyst (Day 10, 38 vs 12%) formation than Unt-Vero cells. These rates were comparable to those obtained with BOECs; blastocyst hatching was slightly higher following co-culture with Mit-Vero cells (36%) than BOECs (29%). Blastocyst formation was similar in ECS- vs BSA-supplemented medium; however, hatching was greatest (37%) during co-culture in medium +10% ECS. While the addition of LIF throughout the co-culture period was ineffective, addition of the cytokine beginning at Day 4 slightly increased blastocyst formation rates. Evaluation of LIF secretion using ELISA revealed detectable levels of the cytokine in Mit-Vero-conditioned medium (50 pg/10 5 cells); this may explain the minimal influence of exogenous LIF during embryo co-culture. Mit-Vero cells provided comparable support of bovine embryo development when used even up to 2 wk after establishment as monolayers. In conclusion, Mit-Vero cells provide a readily-available, safe and easy-to-use co-culture method which is at least as supporting of bovine embryo development as BOECs. One contribution of these cells may be secretion of the cytokine LIF.

Chromosome configuration during in vitro maturation of goat, sheep and buffalo oocytes

The competence of meiotic chromosome configuration at the time of co-culture of oocytes with spermatozoa is an essential prerequisite for successful in vitro fertilization (IVF). Although this technology has been used in several livestock species, various intrinsic and extrinsic factors affecting the high repeatablity of IVF have yet to be understood. The present study was conducted to determine the appropriate time for coculture of oocytes and spermatozoa in order to optimize the fertilization rate in sheep, goats and buffalo. Oocytes were collected from the ovaries of slaughtered animals. The oocytes were divided into 10 groups and cultured for maturation in TCM-199 supplemented with estrous cow serum for different durations at 38.5 · 0.5|C in a CO 2 incubator. Sheep and goat oocytes were removed from culture medium after 0,6,12,22,24,26,28,30,32 and 36 and buffalo oocytes after 0,6,12,16,20,22,24,26,28, and 36 h. The oocytes were treated with hypotonic solution (0.75M KCl) and fixed in Carony's fixative on glass slides. The fixed oocytes were stained with Giemsa solution, and the meiotic chromosomes were evaluated under a compound microscope at x1000 magnification. Observations were recorded on a total of 1328 oocytes (sheep, 409; goat, 727 and buffalo, 192). The sequential configurations of diffused chromatin, pachytene, diplotene (along with nucleoli), diakinesis and metaphase II (MII) were analyzed at different durations of culture. Control oocytes (fixed at 0 h without incubation) were mostly at the pachytene stage, and as the duration of culture increased the instances of diplotene, diakinesis and finally MII increased. Oocytes at the MII stage of meiosis are known to be at the optimal stage of development for co-culture with spermatozoa and successful in vitro fertilization. On the basis of sequential configuration of chromosomes, it was found that the optimal duration of in vitro maturation of oocytes is 32, 30 and 24 h for sheep, goats and buffalo, respectively.

Growth hormone enhances fertilizability of in vitro matured bovine oocytes

The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with frozen-thawed semen separated by swim-up and treated with heparin was carried out to determine the effects on fertilization of 1) increasing sperm concentrations (1× 106, 5 × 106 and 1 × 107sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Forty-nine percent of the collected oocytes (335683) showed a compact cumulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%,3239) and ECS (87.5%,5664) groups, with no statistically significant difference. In Experiment 2, the percentage of normal fertilization (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%,431; 15.2%,959 and 15.5%,958). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 541 with EMS vs 12.9%, 431 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 1443 vs 12.2%, 541; P < 0.05). The incidence of polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumulus removal were further cultured for 72 h two embryos, one at the 2-cell stage and the other at the 4-cell stage, could be obtained. These results indicate that, in the horse, the cumulus can be partially removed to increase the fertilization of compact-cumulus oocytes recovered during the breeding season using frozen-thawed, heparin-treated semen.

The effect of oocyte preparation on the embryo cleavage rate after in vitro fertilization in the pig

We conducted a cytogenetic study of bovine parthenotes derived from oocytes matured and cultured in vitro. In vitro maturation was carried out by culturing follicular oocytes for 24 h in TCM199 supplemented with estrous cow serum (ECS) and hormones at 39 °C in 5% CO2. Matured oocytes were incubated for 20 h in sperm TALP without the addition of spermatozoa, after which they were cultured in maturation droplets for 48 to 72 h. Spontaneous activation occurred in 9.5% of the matured oocytes. Cytogenetic analysis of 24 parthenotes revealed that 62.5% exhibited a normal, diploid chromosome complement. The remaining 37.5% had various ploidy anomalies: haploidy (25%), triploidy (4.2%) and tetraploidy (8.3%). Parthenotes exhibited different developmental stages. The number of blastomeres ranged from 2 to 8 within a parthenote. Only 1 parthenote was comprised 9 to 16 cells. The results showed that spontaneous parthenogenetic activation which occurs in an IVM/IVF system may interfere with embryo production efficiency.

Effects of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development.

In vivo, oocytes mature while bathed in preovulatory follicular fluid, so we studied maturation of oocytes in vitro in media supplemented with such fluid. Bovine oocytes were obtained from 2-to 6-mm follicles from slaughterhouse ovaries and were matured for 24 h in tissue culture medium 199 + 10% estrous cow serum supplemented with bovine follicular fluid (BFF) obtained from a preovulatory follicle either before (0 h) or 8 or 20 h after a GnRH-induced LH surge. After in vitro fertilization, oocytes matured in 10% or 40% 20-h BFF had higher cleavage rates (p < 0.05) than those matured in 10% or 40% 0-h BFF (80% and 86% vs. 46% and 51%, respectively); oocytes matured in 8-h BFF were intermediate. A greater percentage of oocytes showed cumulus expansion with 20-h BFF than with 0-h BFF (p < 0.05). In experiment 2, more (p < 0.05) embryos from oocytes matured in 20% or 40% 20-h BFF developed to blastocysts after culture of embryos compared to those matured in 20% or 40% 0-h BFF (29% and 29% vs. 7% and 14%, respectively) or those matured in no BFF (12%). Embryos from oocytes matured in a mixture of 0-h and 20-h BFF developed to the blastocyst stage similarly (27%) to those matured in 20-h BFF, suggesting that positive factors in 20-h BFF overcame any inhibitory effect of 0-h BFF. To summarize, bovine oocytes matured with BFF collected 20 h after LH surge result in higher cleavage rates after in vitro fertilization and more blastocysts than those matured in 0-h BFF. When the factors responsible are identified, they might be used to enhance maturation of oocytes in vitro.

Regulação da maturação e expansão do cumulus pelas proteínas quinase A e C em oócitos bovinos

The present study was conducted to determine the effect of protein kinase-C (PK-C) and the possible interaction with the protein kinase-A (PK-A) in the resumption of meiosis, nuclear maturation and cumulus expansion in bovine oocytes. Also, the effect of PK-C stimulation in cytoplasmic maturation and embryo development was investigated. In a pre-experiment, oocytes in germinal vesicle (GV), metaphase I (MI) and metaphase II (MH) stages were fixed in acetic acid: methanol (1:3) either on slides (FLL) or in petri dishes (FPL) and stained with 1% of lacmoid in phosphate buffered saline (PBS). Also, the oocytes were fixed on slides immersed in acetic acid: methanol (1:3) and stained with Giemsa (FLG). In the experiments that studies the role of second messengers on oocyte maturation, the oocytes were aspirated using a vacuum pump from 2-8mm follicles, from ovaries obtained in a slaughterhouse, and were cultured in modified TCM 199 medium containing 25mM hepes, 2.2mg/ml sodium bicarbonate and 3mg/ml bovine serum albumin (BSA) at 39°C under an atmosphere of 5% CO2 in air and saturated humidity. The following experiments were accomplished: l) the oocytes were stimulated by different second messengers during 7 or 18h to evaluate nuclear maturation (at 7h and 18h) and cumulus expansion (at 18h); 2) oocytes were stimulated in the presence of fetal bovine serum, and the effects of PK-A and PK-C in the regulation of nuclear maturation were investigated; 3) oocytes were cultured during a period of 24h in the presence of estrous cow serum suppiemented with LH (1.5mg/ml), FSH and LH (0.5mg/ml and 1.5mg/ml) or PMA (active phorbol ester, stimulator of PK-C; l00nM), for investigating cleavage and embryo development rates. In the pre-experiment, the GV stage was accurately identified only with the FLL technique. However, FPL and FLG procedures were more efficient for oocytes in the MI and MII stages. PMA and l .2-dioctanoyl-sn-glycerol (DiC8) stimulated meiotic resumption and nuclear maturation in a dose dependent manner (0-l00nM and 0-l0mM, respectively). The treatment with forskolin (FK) significantly inhibits resumption of meiosis and decreases the percentage of oocytes in the MH stage, but PK-C stimulation reversed this inhibitory effect. Forkolin stimulated cumulus expansion in the presence and absence of PMA; however, PMA had only a slight effect in cumulus expansion. There were no significant differences in cleavage and embryo development among treatment groups. The results suggest that PK-C is involved in the regulation of oocyte maturation in the bovine, stimulating both the resumption of meiosis and nuclear maturation. Also, PK-C suppresses the inhibitory effect of PK-A on oocyte nuclear maturation. In regarding to cumulus expansion, PK-C and PK-A are involved in the regulation of cumulus cells mucification.

Effects of platelet-derived growth factor (PDGF) on the in vitro production of bovine embryos in protein-free media

The purpose of our experiments was to explore the effects of platelet-derived growth factor (PDGF)-supplementation at the various steps of in vitro production of bovine embryos using protein-free media. Cumulus-oocyte-complexes (COC) were collected by slicing abattoir ovaries and then dividing the COC into 2 morphological categories. After maturation for 24 h in TCM-199 supplemented with hormones and either 20% estrous cow serum (ECS) or 1 mg/ml polyvinyl-alcohol (PVA), oocytes were co-incubated for 19 h with frozen/thawed spermatozoa from bull of proven fertility. The semen was diluted in Fert-Talp supplemented with heparin, hypotaurine and epinephrine and either 6 mg/ml bovine serum albumin (BSA) or 1 mg/ml PVA. Presumptive zygotes were transferred into embryo culture medium containing either 20% ECS or 1 mg/ml PVA for a total of 10 d. The PDGF was added at concentrations of 1, 10 or 100 ng/ml to the maturation medium (Experiment 1), fertilization medium (Experiment 2) or culture medium from Day 1 on (Experiment 3), respectively, or at 1 ng/ml PDGF to both the fertilization and culture medium from Day 3 on (Experiment 4), with each medium supplemented with PVA. Oocytes/embryos incubated in the absence of PDGF in media supplemented with either ECS or PVA served as controls. An average of 20 COC was incubated in 1 droplet under silicone oil, and each experiment contained 4 to 6 replicates. No significant differences were found among the various concentrations of PDGF, nor did PDGF-supplementation during maturation (Experiment 1) or embryo culture on Day 1 (Experiment 3) significantly affect development of oocytes/embryos (34.7 ± 3.5 to 40.4 ± 2.5% morulae, 11.9 ± 2.4 to 18.8 ± 2.5% blastocysts; and 23.2 ± 2.3 to 27.5 ± 3.4% morulae, 11.5 ± 2.6 to 12.7 ± 2.3% blastocysts, respectively; x ± SEM). In the presence of 10 ng/ml PDGF in the fertilization medium development to morulae and blastocysts was similar to that of the ECS-group, and was higher (P < 0.05) than that of the PVA-control (ECS: 32.1 ± 4.6 and 13.8 ± 2.7%; PVA: 17.5 ± 0.8 and 6.1 ± 1.3%; PDGF: 30.6 ± 3.0 and 14.0 ± 2.2%, respectively). Development to morulae/blastocysts was increased, and was at the same level as in the ECS-group when the fertilization and/or embryo culture medium on Day 3 contained PDGF compared with the PVA-control group (morulae: ECS 25.3 ± 4.4%, PVA 13.9 ± 2.2% [P < 0.05], PDGF 16.7 ± 3.2 to 19.1 ± 1.1%; blastocysts: ECS 5.3 ± 2.1%, PVA 5.0 ± 1.7%, PDGF 7.1 ± 1.6 to 9.1 ± 1.7%, respectively). These results indicate that under our laboratory conditions PDGF can elevate low rates of development and the addition of PDGF to the fertilization medium enhances bovine preimplantation embryonic development. Thus, PDGF can be potentially an important factor in a completely defined medium to substitute the effects of serum.

The incidence of bovine diploid oocytes matured in vitro

The present work describes a cytogenetic study of in vitro matured bovine oocytes to determine the proportion of unreduced oocytes carrying the diploid number of chromosomes. Studied oocytes were derived either from a pool of oocytes collected from several different donors, or from oocytes collected separately from individual donors. In vitro maturation was performed by culturing immature oocytes for 24 h in TCM199-medium supplemented with estrous cow serum and hormones at 39 °C in 5% CO 2. Chromosomal complement of in vitro matured oocytes was studied by Giemsa-staining and produced analyzable results in approximately 60% of the cases. The results revealed that approximately 75% of oocytes had matured to the MII stage in both groups of oocytes studied. Of these MII oocytes, 11 and 12.4% (from the oocyte pool or from individual cows, respectively) contained the diploid set of chromosomes. The occurrence of diploid MII oocytes was not quite uniform among donors: 40.5% of all cows produced one, 18.9% produced two and 2.7% (one cow) produced three diploid MII oocytes. However, a positive relationship between the number of MII oocytes in general and diploid MII oocytes among individual donors was not found. The possible factors that may lead to the formation of diploid MII oocytes observed under IVM procedures are discussed. The results of this study showed a higher incidence of diploid oocytes in cattle than previously reported.

Effects of Media, Serum, Oviductal Cells, and Hormones During Maturation on Bovine Embryo Development In Vitro

Our objective was to optimize in vitro maturation conditions of bovine oocytes as assessed by embryo development. In Experiment 1, cumulus-oocyte complexes were matured in either M-199 or RPMI-1640. Each medium was supplemented with an antibiotic-antimycotic solution (1%) and estrous cow serum (20%). Cumulus cell expansion after 24h was greatest for cumulus-oocyte complexes matured in RPMI-1640. Morulae development on d 7 was greater (21.1%) for oocytes matured in M-199 than for oocytes that matured in RPMI-1640 (9.6%). In Experiment 2, cumulus-oocyte complexes were matured in M-199 supplemented with antibiotic-antimycotic solution (1%). Main effects were serum type (20%; estrous cow serum vs. superstimulated estrous cow serum) and coculture (with or without bovine oviductal epithelial cells). The percentage of oocytes developing into blastocysts (d 9) was higher for oocytes matured in estrous cow serum regardless of coculture. In Experiment 3, effects of estradiol-17β (0, 1, and 2μg/ml) and equine LH (0, 10, 20, and 30μg/ml) on cumulus cell expansion and development after fertilization were determined. Cumulus cell expansion and blastocyst development decreased with estradiol-17β in the maturation medium, but LH in the medium enhanced expansion of cumulus cells and blastocyst development.

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 μg/ml gentamycin, 10 μg/ml FSH, 1 μg/ml estradiol-17β). 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.

In vitro maturation and fertilization of equine oocytes recovered during the breeding season

The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with frozen-thawed semen separated by swim-up and treated with heparin was carried out to determine the effects on fertilization of 1) increasing sperm concentrations (1× 10 6, 5 × 10 6 and 1 × 10 7sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Forty-nine percent of the collected oocytes ( 335 683 ) showed a compact cumulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%, 32 39 ) and ECS (87.5%, 56 64 ) groups, with no statistically significant difference. In Experiment 2, the percentage of normal fertilization (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%, 4 31 ; 15.2%, 9 59 and 15.5%, 9 58 ). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 5 41 with EMS vs 12.9%, 4 31 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 14 43 vs 12.2%, 5 41 ; P < 0.05). The incidence of polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumulus removal were further cultured for 72 h two embryos, one at the 2-cell stage and the other at the 4-cell stage, could be obtained. These results indicate that, in the horse, the cumulus can be partially removed to increase the fertilization of compact-cumulus oocytes recovered during the breeding season using frozen-thawed, heparin-treated semen.