Expression of DNA methyltransferase genes in four-cell bovine embryos cultured in the presence of oviductal fluid.

The possible epigenomic effect of oviductal fluid on expression of DNA methyltransferase (DNMT) genes was examined in early bovine embryos (4-cell stage). Real-time quantitative PCR was performed to determine the relative expression of DNMT1, DNMT3a and DNMT3b transcripts in embryos cultured in vitro in the presence or absence of oviductal fluid. Expression of DNMT1 significantly increased when cultured with oviductal fluid, whereas DNMT3a and DNMT3b transcripts were unaffected by the addition of oviductal fluid. These results may help reveal the role of oviductal factors in the regulation of DNMT expression.

Effects of sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts.

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.

Male and female effects on the in vitro production of bovine embryos.

A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3-93.4%) and blastocysts on day 7 (6.9-51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F-value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme.

Effects of growth hormone on female reproductive organs.

During the last decade many experiments have been performed to study the effects of growth hormone (GH, somatotropin) on reproductive functions. Most of the studies found only slight or no effects of GH treatment, both on the oestrous cycle and on gonadotropin, progesterone. or oestrogen serum levels. In GH-treated animals, elevated levels of insulin-like growth factor I and GH in the serum could be correlated with an increased number of small (< 5 mm in diameter) ovarian follicles, possibly as a consequence of a reduction of apoptosis and follicular atresia. There is still controversy over the effects of GH on in vivo and in vitro embryo production and on the gestation period. Recent studies produced some evidence that GH-receptor is expressed in ovarian tissue, implying a direct role for GH in the ovary.

Developmental capacity in vitro of prepubertal oocytes.

A 2-year comparative study was carried out to evaluate the effect of ovary size, follicle size and oocyte quality of 3-month-old Simmental calves and the efficiency of using calf ovaries in an in vitro fertilization (IVF) programme. We evaluated the effects of different concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta (E-17beta) in the maturation medium on the in vitro development of calf oocytes into morula and blastocysts. The proportion of recovered oocytes (62.1%; 42.8%; 25.3%) and the percentage of good quality cumulus oocyte complexes (84.2%: 59.8%; 45.9%) decreased significantly (P < 0.01) with decreasing ovary size (L, M and S). The rates of two or more cells on Day 2 and of blastocysts on Day 7 and Day 9 were significantly lower (P < 0.01) for calf oocytes (61.5%; 18.9%: 15.9%) compared with those from sexually matured females (70.1%: 32.3%; 22.2%). Calf oocytes. matured in medium supplemented with 20 microg/ml or 10 microg/ml FSH plus 2 microg/ml E-17beta had higher rates of cleavage on Day 2 (64.1% and 64.7%) and blastocysts on Day 7 (24.5% and 22.4%) than the control supplemented with 10 microg/ml FSH (55.6% and 19.2%, respectively). Groups supplemented with 20 microg/ml FSH plus 2 microg/ml E-17beta and 10 mg/ml plus 4 mg/ml E-17beta showed a significantly lower developmental rate of blastocysts on Day 7 (14.6% and 14.5%). High concentrations of E-17beta (4 microg/ml) resulted in a significantly lower development of blastocysts on Day 9 (8.1%) and hatched blastocysts on Day 13 (3.5%) (P < 0.01). We conclude that the proportion of calf oocytes obtained from immature animals and their suitability for IVF are lower than those of cows. Thus, the use of oocytcs from sexually immature females would decrease the relative efficiency of IVF programmes. Supplementation with high concentrations of FSH can improve the maturation and developmental capacity of oocytes from prepubertal calves.

Pregnancy rate after ultrasound-guided follicle aspiration in nonlactating cows from different breeds.

Thirty-six cows (21 Simmental, 5 Holstein-Frisian, 5 Brown Swiss and 5 Charolais) with high genetic superiority were punctured by ultrasound-guided follicle aspiration within the last 18 months for 503 times under equal conditions. Follicle aspiration was done twice per week. Most of the donor cows suffered from several disturbances of fertility. On average, 5 oocytes per session were collected. After in vitro maturation (IVM), fertilisation (IVF) and culture (IVC), 0.8 embryos per puncture session were transferred. After evaluation of the embryos by morphological criteria, these embryos were transferred to heifers that were oestrus synchronised (2.0 ml Estrumate i.m.) seven days after onset of oestrus. On day 21 after onset of oestrus the progesterone level in plasma was determined by radioimmunoassay. The pregnancy control was performed by ultrasound on day 35. After transfer of 397 embryos (to synchronised heifers), 125 pregnancies were established. Comparison of the different breeds and donor cows revealed significant differences in the number of oocytes recovered, embryos produced and pregnancies established. Transfer of embryos with insufficient morphological quality resulted in increased rates of embryonic loss between day 21 and day 35.

Effect of insulin-like growth factor I (IGF-I) at high concentrations on blastocyst development of bovine embryos produced in vitro.

This study was carried out to determine the effects of oestrous cow serum containing insulin-like growth factor I (IGF-I) and supplementation with recombinant IGF-I on subsequent development of bovine embryos produced in vitro. When culture medium was supplemented with oestrous cow serum containing 34.8 ng endogenous IGF-I ml-1, more embryos (P < 0.01) developed to blastocysts by day 9 and more blastocysts hatched on day 13 after insemination (P < 0.01) than in the control group. The effect of the addition of 10, 50 and 100 ng IGF-I ml-1 to culture media containing oestrous cow serum and granulosa cells was also evaluated. Supplementation with 10 ng IGF-I ml-1 did not improve embryo development at any stage. The addition of 50 and 100 ng IGF-I ml-1 did not affect development during the first three cell divisions. However, on day 7 these groups yielded a higher embryo rate than did the control group. Similarly, the proportion of blastocysts on day 9 was enhanced. The addition of 100 ng IGF-I ml-1 also increased the proportion of blastocysts. These data suggest that IGF-I at high concentrations accelerates the development to the blastocyst stage by shortening the transition from the morula to the blastocyst stage. The addition of 100 ng IGF-I ml-1 increased the proportion of hatched blastocysts on day 13. The addition of oestrous cow serum and IGF-I to TCM 199 free of granulosa cells did not increase the proportion of embryos on day 7. However, the progress to blastocysts and hatched blastocysts on days 9 and 13 was significantly lower (P < 0.05). The addition of IGF-I to culture medium without oestrous cow serum but with granulosa cells resulted in significantly lower embryo development than in the control group or in the group supplemented with oestrous cow serum and IGF-I (P < 0.01). The results support the hypothesis that culture media containing high concentrations of IGF-I combined with oestrous cow serum and granulosa cells can improve the development of embryos produced in vitro.

Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: effect of developmental stage.

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 +/- 4.1) rather than nuclei from morulae (9.8 +/- 5.5) or blastocysts (6.3 +/- 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 +/- 5.9) than did morulae (9.3 +/- 3.2) and blastocysts (3.3 +/- 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts.

Fertilization of bovine oocytes induced solely with combined laser microbeam and optical tweezers.

PURPOSE: Our purpose was to show that fertilization of oocytes can be obtained solely by laser light-mediated manipulation of gametes. METHOD: A small channel was drilled into the zona pellucida of bovine oocytes using an ultraviolet (UV)-laser microbeam. Highly diluted cattle sperm were not able to fertilize the laser drilled oocytes. RESULTS: Fertilization was achieved only when three to five cattle sperm were trapped with optical tweezers and inserted directly through the laser drilled hole into the perivitelline space. After 20 hr, 3 of 79 (3.8%) oocytes revealed two pronuclei and a sperm tail within their cytoplasm. Cattle sperm are difficult to catch. Therefore, the gametes had to remain for about 20 min in room atmosphere, which might be the reason for the low fertilization results. CONCLUSIONS: The results indicate that a combined UV-laser microbeam and optical tweezers trap can be used successfully for "noncontact" microinsemination procedures.

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning.

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25-34), intermediate (40-55), and high (60-81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50-83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40-55) or high (60-81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40-55 cells) and 41% versus 25% (60-81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning.

Spinal somatosensory evoked potentials following segmental sensory stimulation. A direct measure of dorsal root function.

Dorsal root function cannot presently be measured directly. The H-reflex is an indirect measure of dorsal root function but only for the S1 root. Spinal somatosensory evoked potentials (SEPs) following dermatomal stimulation of the legs have the potential of providing direct data reflecting dorsal root function but have not been reliably recorded in normal subjects. We have developed a reliable technique for recording SEPs at the lumbar root entry zone following segmental sensory stimulation of the legs. The saphenous, superficial peroneal, and sural nerves were stimulated representing the L3/L4, L5 and S1 roots respectively. Reproducible responses (N-wave) were recorded over the lumbar spine in all 60 normal limbs examined. The N-wave peak latency was significantly correlated with lower limb length. The conduction velocities from the stimulation sites to the lumbar spine were similar to published values for peripheral conduction velocities in these nerves. The mean inter-limb latency differences for the N-wave peak were: L3/L4 0.61 msec; L5 0.35 msec; and S1 0.57 msec. The mean N-wave amplitudes were: L3/L4 0.11 microV; L5 0.28 microV; and S1 0.23 microV. This technique is a direct measure of dorsal root integrity. Unlike scalp recorded SEPs, the lumbar N-wave is not state-dependent and is unaffected by lesions within the brain and rostral cord.