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Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.

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 (P0.05) 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% [P0.05], 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.

This study examined the role of protein supplementation at the various steps of the in vitro production of bovine embryos derived from two different morphological categories of COC. The basic medium was TCM 199 and was supplemented with hormones during maturation in vitro and either estrous cow serum (ECS), bovine serum albumin (BSA) at various concentrations or polyvinyl-alcohol (PVA). Fertilization in vitro was carried out using frozen-thawed semen or one bull in Fert-talp containing heparin, hypotaurin and epinephrine and either 6 mg/ml BSA or 1 mg/ml PVA. In vitro culture up to the blastocyst stage was performed in TCM 199 supplemented with either ECS, BSA or PVA. The first experiment investigated the influence of different medium-supplements (ECS, BSA or PVA) on nuclear maturation and revealed no significant differences among treatment groups nor between categories of COC (63.9% to 74.9% and 48.9% to 77.0%, respectively). The time course of in vitro fertilization was elucidated in Experiment 2 in medium supplemented with either protein or PVA during maturation and fertilization. Penetration was not affected (70.9% to 79.3% penetration 12 h after onset of oocyte-sperm-co-incubation), but formation of pronuclei was decreased (P0.05) 12 and 19 h after onset of oocyte-sperm-co-incubation and was retarded in medium supplemented with PVA (12 h: 63.8 vs 21.4 %; 19 h: 57.5 vs 20.8 %, respectively) while cleavage was not affected. In Experiment 3, six treatment groups were formed in which the two different morphological categories of cumulus-oocyte-complexes (COC) were incubated in basic medium supplemented with 1) ECS during maturation and embryo culture and BSA during fertilization; 2) PVA during maturation and embryo culture, fertilization medium with PVA; 3) PVA during maturation and embryo culture, fertilization medium with BSA; 4) BSA (1 mg/ml) during maturation, fertilization and embryo culture; 5) BSA (6 mg/ml) during maturation, fertilization and embryo culture; and 6) BSA (10 mg/ml) during maturation, fertilization and embryo culture. The rates of cleavage and the development to morulae or blastocysts did not differ (P0.05) among treatment groups and between both categories of COC and were showing a high degree of variability (cleavage 54.0% to 65.1% and 41.3% to 55.7%, respectively; morulae 25.3% to 53.0% and 26.0% to 51.2%, respectively; blastocysts 5.4% to 24.7% and 0.6% to 20.3%, respectively). Parthenogenetic activation only rarely occurred in medium containing PVA throughout all steps of in vitro production of bovine embryos (Experiment 4) and led to early cleavage stages (8%), but no development to morula- or blastocyst-stages was observed. It is concluded that 1) formation of pronuclei was retarded in medium lacking protein-supplementation, indicating that BSA is required for regular fertilization in vitro and 2) under our experimental conditions, protein-supplementation is not necessary for maturation and development up to the blastocyst stage in vitro.

Ultrasound-guided follicular aspiration was performed on 29 Holstein-Friesian cows/heifers twice weekly at 3- to 4-d intervals over a period of 2 consecutive estrous cycles (total 42 d). For visualization of the ovaries and guidance of the aspiration needle, a 6.5 MHz fingertip probe on a 62 cm probe carrier was inserted into the vagina. The disposable aspiration needle was connected to a permanent rinse tubing system, thus ensuring minimum death of oocytes in the aspiration processs. After penetration of the vaginal wall, the needle was inserted into a follicle of the rectally fixed ovary. Cumulus oocyte complexes (COC) were aspirated at a pressure of 100 mm Hg. In the first experiment, the effect of an additional gonadotropin treatment 4 d prior to aspiration was investigated in 8 lactating cows. Following FSH-treatment, the number of aspirated follicles was higher (P < 0.05) than in the nontreated animals (10.6 +/- 0.7 vs 8.9 +/- 0.5). The number of recovered COC (7.0 +/- 0.6 vs 5.8 +/- 0.5), the recovery rate (COC per aspirated follicle) (66.6% vs 65.4%), the percentage of viable COC (56.8% vs 52.1%), the cleavage rate upon in vitro maturation and in vitro fertilization (56.7% vs 59.8%) as well as the rate of morula/blastocyst formation (3.8% vs 2.9%) were similar in both groups. In the second experiment, follicles were aspirated in 4 lactating cows, 6 dry cows, 4 pregnant cows (first 35 d of pregnancy), and 4 heifers. The average number of aspirated follicles and recovered COC was higher (P < 0.05) in the first 2 groups (10.6 +/- 0.6 and 9.3 +/- 0.7 follicles; 7.2 +/- 0.5 and 6.9 +/- 0.7 oocytes) than in trie 2 other treatment groups (7.3 +/- 0.5 and 8.1 +/- 0.5 follicles; 5.0 +/- 0.4 and 5.7 +/- 0.5 oocytes). The percentage of viable COC was higher (P < 0.05; 68.3%) in lactating animals than in all the other groups (49.7, 52.5 and 57.4%, respectively). Similarly, upon in vitro fertilization, cleavage rate was higher (P < 0.05; 63.4%) in lactating cows than in the other groups (43.7, 50.5, 55.1%, respectively). A total of 21.5, 22.7, 11.9 and 13.5%, respectively, in the 4 groups of the in vitro fertilized oocytes reached the morula and blastocyst stages. After transfer of a total of 48 embryos 22 pregnancies (45.8%) were established as detected on Day 65. We conclude that 1) repeated aspiration of viable COC at short intervals is possible, 2) additional FSH-treatment does not increase oocyte yields, and 3) viable blastocysts can be produced from cattle at various reproductive phases irrespective of the reproductive phase.

A total of 917 cumulus-oocyte complexes were used in this experiment. In Group VI the cumulus-oocyte complexes were matured and fertilized in vitro. After an initial 48 hours culture in microdrops they were transferred to a granulosa cell monolayer. Development to morulae/blasto-cysts was evaluated 8 days after in-vitro fertilization. The treatment for Group I consisted of 50 ng/ml insulin-like growth factor I (IGF-I) added to in-vitro maturation media. Group II received IGF-I to in-vitro culture on the granulosa cell monolayer. In Group III of IGF-I was added to in-vitro maturation media and in-vitro culture on the granulosa cell monolayer. Group IV received IGF-I to in-vitro culture and was cultured the whole time without a granulosa cell monolayer. Group V received IGF-I to in-vitro maturation and were cultured in-vitro without a granulosa cell monolayer. After supplementation of in-vitro maturation media with IGF-I, 52, 49 and 51% of the cumulus-oocyte complexes in Groups I, III and V did not show cumulus expansion, whereas in Group VI (control) only 1% of the cumulus-oocyte complexes did not expand (P

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P0.001), 300 x 10(6) liquid conserved semen (49.0%, P0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.

This study investigated the effects of a purified follicle stimulating hormone (FSH) preparation supplemented with three different amounts of bovine luteinizing hormone (bLH) and a commercially available FSH with a high LH contamination on superovulatory response, plasma LH and milk progesterone levels in dairy cows. A total of 112 lactating Holstein-Friesian crossbred dairy cows were used for these experiments; the cows were randomly assigned to treatment groups consisting of purified porcine FSH (pFSH) supplemented with bLH. Group 1 was given 0.052 IU LH/40 mg armour units (AU) FSH (n = 6); Group 2 was given 0.069 IU LH (n = 32); Group 3 received 0.423 IU LH (n = 34); while Group 4 cows (n = 36) were superovulated with a commercially available FSH-P. This compound appeared to contain 8.5 IU LH/40 mg AU FSH according to bioassay measurement. All animals received a total of 40 mg AU FSH at a constant dose twice daily over a 4-d period. Levels of milk progesterone and plasma LH were determined during the course of superovulatory treatment. The Group 1 treatment did not reveal multiple follicular growth, and no embryos were obtained. Superovulation of Group 3 cows resulted in significantly (P

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 ( 8 15 ), 46.2 ( 6 13 ), and 47.5% ( 9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

This study investigated the accuracy of a commercially available rapid milk progesterone (P(4)) assay (RMPA) and its usefulness for the screening of potential donor cows prior to superovulatory treatment. Superovulation was induced in 90 lactating Holstein-Friesian crossbred dairy cows with twice daily injections over a 4-day period for a total of 40 mg follicle stimulating hormone (FSH-P), starting 9 to 13 d post estrus. Prior to induction of superovulation, a milk sample was collected and assayed for a P(4) level using the RMPA. The test determines P(4) by a simple visual color inspection of the respective sample, which is compared to a standard containing 10.5 ng/ml of P(4). All animals were divided into six groups according to the color intensity of their sample; three groups had a lower level, one group had an equal level and two groups had a higher P(4) level than the standard. Results of the semiquantitative RMPA were verified by a quantitative enzymeimmunoassay (EIA). Samples evaluated as equivalent to the standard had a mean P(4) level of 10.7 +/- 1.3 ng/ml (x +/- SEM). In total, P(4) levels differed (P

The influence of fast freezing and thawing on bovine embryos at different stages of development was investigated. A total of 20 day-7 embryos and 37 day-8 embryos were thawed and classified morphologically before being transferred nonsurgically to synchronized recipients. Ninety percent ( 18 20 ) of the day 7 embryos (late morulae and blastocysts) were classified as transferable and a pregnancy rate of 52.9% ( 9 17 ) was obtained with these embryos. Seventy eight percent ( 29 37 ) of the day 8 embryos (expanded blastocysts) were classified as transferable, but only 24.1% ( 7 29 ) of these embryos resulted in pregnancy. The best pregnancy rate was obtained with the blastocysts of day 7 ( 5 8 or 62.5%), which compares favorably with that of freshly collected embryos. It is suggested that the low pregnancy rate found in the day 8 embryos is related to ultrastructural damages of the desmosomes and tonofilaments within the trophoblast layer, which results in a disturbance of the normal hatching process.

The effects of a one-step addition of 1.4 M glycerol (method A) upon morphological appearance and developmental capacity of frozen/thawed day 7 bovine embryos were investigated and compared to a standard stepwise addition of 1.0 M glycerol (method B). With method A, the percentage of intact embryos (classified as excellent, good and poor) was 95.3% (61 out of 64) without differences between morulae (96.5%) and blastocysts (94.4%). With method B, the percentage of intact embryos was 83.0% (44 out of 53). The percentage was similar for blastocysts (89.2%) and significantly (p < 0.05) lower for morulae (68.8%) when compared to method A. The percentage of embryos with a damaged zona pellucida was considerably increased with method A (26.6%) when compared to method B (13.2%). The proportion of embryos with excluded blastomeres was similar in both methods (21.9% method A, 17.0% method B). With method A, pregnancy rates after nonsurgical transfer were 51.0% (25 out of 49) and were better than with method B (40.5%; 15 out of 37). Embryos with a damaged zona pellucida resulted in a high pregnancy rate of 66.7% (8 out of 12). A pregnancy rate of 52.9% (10 out of 17) was obtained with embryos showing some excluded blastomeres. Thus, a one-step addition of 1.4 M glycerol facilitates and accelerates the process of embryo cryopreservation and is compatible with high pregnancy rates. Damage of the zona pellucida does not impair further development of frozen/thawed bovine embryos provided blastomeres are intact.

A total of 71 synchronized dairy heifers (Holstein Friesian × German Black Pied) were used as recipients of seven-day old frozen/thawed bovine embryos. Plasma progesterone concentrations and corpus luteum quality on the day of nonsurgical transfer (= day 7) were determined and related to pregnancy rates or estrus intervals in nonpregnant recipients. A total of 32 recipients (45.1 %) maintained pregnancy; 39 recipients (54.9 %) did not. No significant differences could be detected between progesterone levels in recipients that remained pregnant (3.14 ± 0.24 ng/ml; x ± SEM ) and those that did not maintain pregnancy (3.23 ± 0.28 ng/ml). Optimal progesterone levels were between 2 and 5 ng/ml coinciding with a pregnancy rate of 51.1 % ( 24 47 ). Pregnancy rates apparently were decreased when progesterone levels were below 2 ng/ml (35.3 %; 6 17 ) or above 5 ng/ml (28.6 %; 2 7 ). Hence, optimal progesterone levels were identical to those for freshly collected embryos reported previously by Remsen et al. (1). Bovine corpus luteum quality graded by rectal palpation was related to some extent to progesterone levels but not to pregnancy rates. Out of 39 nonpregnant recipients seven animals (17.9 %) with a mean plasma progesterone level of 3.76 ± 0.72 ng/ml showed an extended estrus interval of more than 55 days, probably indicating early embryonic mortality. Progesterone levels did not significantly differ between nonpregnant recipients with estrus intervals of various length. Plasma progesterone levels at the time of transfer are of limited diagnostic value for screening recipients prior to transfer of frozen/thawed embryos.