S.J. Dieleman

Factors affecting oocyte quality and quantity in commercial application of embryo technologies in the cattle breeding industry

With the introduction of multiple ovulation, embryo recovery and transfer techniques (MOET) plus embryo freeze-thaw methods in the early 1980s, the breeding industry has the tools in hand to increase the number of calves from donors of high genetic merit. In the early 1990s, the introduction of ovum pick-up followed by in vitro embryo production (OPU-IVP) opened up even greater possibilities. Using these technologies, we challenge biological mechanisms in reproduction. Where normally one oocyte per estrous cycle will develop to ovulation, now numerous other oocytes that otherwise would have degenerated are expected to develop into an embryo. Completion of oocyte growth and pre-maturation in vivo before final maturation both appear to be essential phases in order to obtain competence to develop into an embryo and finally a healthy offspring. In order to increase oocyte quality and quantity in embryo production technologies, current procedures focus primarily on improving the homogeneity of the population of oocytes with regard to growth and state of pre-maturation at the start of a treatment. In the case of MOET, dominant follicle removal (DFR) before superovulation treatment improves the number of viable embryos per session from 3.9 to 5.4 in cows but not in heifers and a prolonged period of follicle development obtained by preventing release of the endogenous LH surge increases the number of ova but not the number of viable embryos per session. In the case of OPU-IVP, the frequency of OPU clearly affects quantity and quality of the collected oocytes and FSH stimulation prior to OPU every 2 weeks resulted in 3.3 embryos per session. Analysis of 7,800 OPU sessions demonstrated that the oocyte yield is dependent on the team, in particular, the technician manipulating the ovaries. It is concluded that an increased understanding of the processes of oocyte growth, pre- and final maturation will help to improve the efficiency of embryo technologies. However, somewhere we will meet the limits dictated by nature.

Chronology of Apoptosis in Bovine Embryos Produced In Vivo and In Vitro

Abstract The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.

Effects of in vivo prematuration and in vivo final maturation on developmental capacity and quality of pre-implantation embryos

In current in vitro production (IVP) systems, oocytes lack in vivo dominant and preovulatory follicular development, which may compromise pregnancy and viability of calves born. When an oocyte sets off in vivo on the road toward fertilization, it contains numerous transcripts and proteins necessary to survive the first few cell cycles of embryonic development. It is not yet known during which period of development the oocyte builds up the store, possibly primarily during the major growth phase of the oocyte, which is completed at the time a follicle reaches the size of 3 mm. Here, we investigated to what extent the later phases of follicular development, such as prematuration in the dominant follicle before the LH surge and ensuing final maturation in the preovulatory follicle, contribute to oocyte competence and development into viable biastocysts. Recent studies on in vivo vs in vitro oocyte maturation employed oocytes from an identical preovulatory development by applying ovum pick-up (OPU) twice (before and 24 h after the LH surge) in each cow treated for superovulation with a controlled LH surge. The embryo recovery rates at Day 7 of IVC after IVF were similar: 44% (97/219) for in vivo- vs 41% (87/213) for in vitro-matured oocytes, which shows that the natural environment during final maturation is not essential for the mere in vitro development of the prematured oocyte beyond the 8- to 16-cell stage. However, in vivo maturation appeared to contribute to the oocytes quality in a more subtle way, as indicated by a significant increase in the proportion of expanded blastocysts and a more physiological degree of chromosome aberrations of the embryos. In blastocysts derived from in vivo-matured oocytes, 21% of the embryos were mixoploid vs 50% from in vitro-matured oocytes, concomitant with a higher number of cells (96 vs 54 per normal blastocyst). The expression pattern of a set of six developmentally important genes was, however, not significantly altered in blastocysts derived from in vivo-matured oocytes. Certain deviations were observed compared with the levels of entirely in vivo-developed control blastocysts, which suggests that the beneficial effects of in vivo maturation are possibly exerted at initial stages of embryonic development. Prematuration in vivo, occurring in a dominant follicle developing from about 8 mm into the preovulatory follicle, is accompanied by changes in protein synthesis of the cumulus oocyte complex (COC). Presumably, the differentially expressed proteins are involved in equipping the oocyte with further developmental competence. Although we have unraveled some important biochemical and cellular biological features of the oocyte, further research on in vivo processes is essential to improve in vitro embryo production in practice.

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory lh surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.

Consequences of In Vivo Development and Subsequent Culture on Apoptosis, Cell Number, and Blastocyst Formation in Bovine Embryos

Abstract Bovine embryos produced in vitro differ considerably in quality from embryos developed in vivo. The in vitro production system profoundly affects the competence to form blastocysts, the number of cells of the total embryo and of the inner cell mass (ICM), and the incidence of apoptosis. To our knowledge, the effects of different postfertilization regimens before and after completion of the fourth embryonic cell cycle on these aspects have not yet been investigated. In the present study, we assessed the blastulation rate by stereomicroscopy and the cell number of the total embryo, of the ICM, and of the cells with apoptotic changes by confocal laser-scanning microscopy after staining with propidium iodide and TUNEL. Two groups of embryos were developed in heifers, after superovulation, until 45 or 100 h postovulation (po) and, after collection on slaughter, were further cultured in vitro until Day 7 po. A third and fourth group comprised embryos that were produced entirely in vitro or in vivo. The results indicate that passage in vivo of the fourth cell cycle does not prevent acceleration of the formation of the blastocoele in vitro but may be the critical factor contributing to a higher cell number in the total blastocyst and its ICM. The lower quality of in vitro-produced embryos can be attributed to the ICM having less viable cells because of a lower number of cells and a higher incidence of apoptosis that appears to be determined before completion of the fourth cell cycle.

INFLUENCE OF PHYSICAL STRESS ON THE PLASMA-CONCENTRATION OF SERUM AMYLOID-A (SAA) AND HAPTOGLOBIN (HP) IN CALVES

The influence of physical stress on the plasma concentration of the acute-phase proteins serum amyloid-A (SAA) and haptoglobin (Hp) was studied in 10 calves. Two different stress levels were created by housing two groups of five calves, each on different types of floor. The stress level was assessed by studying videotapes of the animals, and, subsequently, by quantifying the problems related with moving across the pens and the time the calves spent lying down and standing. Plasma concentrations of Hp, SAA, aldolase, and cortisol were measured in blood samples obtained by jugular venepuncture. Plasma SAA concentrations were significantly (p < 0.001) elevated in animals housed on the floor type associated with the highest level of physical stress, although the concentrations were within the normal range for healthy adult cattle. Hp concentrations were not elevated. The floor type did not alter the stress related biochemical variables aldolase and cortisol. It is concluded that plasma SAA concentrations rise upon physical stress, whereas Hp concentrations do not change. The absence of a significant difference in aldolase or cortisol concentrations indicates that the difference in the level of neuro-endocrine stress between the animals housed on the two floor types is only minimal. Consequently, SAA is suggested to be a sensitive variable to assess physical welfare in calves.

Regulation and modulation of oocyte maturation in the bovine

Abstract Follicle enclosed oocytes are arrested at the prophase of the first meiotic division. Maturation of oocytes occurs in preovulatory follicles after the preovulatory LH surge concurrently with the final development of the preovulatory follicle. Although there is little doubt about the LH surge as the trigger for the resumption of meiosis, the intrafollicular signals that regulate the arrest before the LH peak and the resumption and progression of meiosis thereafter are still open to question. The available knowledge is predominantly obtained from in vitro maturation studies. This paper presents the state of the art on the action of endocrine and paracrine factors that affect the in vitro maturation of bovine oocytes either directly or mediated by cumulus cells. In particular, the effects of the gonadotropic hormones, growth hormone and several growth factors on nuclear and cytoplasmic maturation are discussed.

Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a qualitative and quantitative milk progesterone test.

We compared three methods for diagnosing early pregnancy in cattle: 1) a trans-rectal ultrasound scan of the uterus, 2) a cow-side enzymeimmunoassay (EIA) milk progesterone test 3) a radioimmunoassay (RIA) milk progesterone test. Scanning of the uterus was performed in 148 cows. These cows were not detected in estrus before scanning, which took place between Days 21 and 33 after insemination (AI). A considerable difference was noted between the reliability of the scannings performed at an early stage (Days 21 to 25) and those performed at a later stage (Days 26 to 33). The sensitivity and specificity of the ultrasound examination between Days 21 and 25 were only 44.8% and 82.3%, respectively, but were 97.7% and 87.8% between Days 26 and 33, respectively. Milk samples were collected on the day of AI. (Day 0) and 21 days later. Samples that were positive in the EIA test always contained more than 1 ng/ml progesterone (P4); however, 20% of the negative EIA samples contained also more than 1 ng/ml P4. Only 59% of the animals showing a negative EIA test on Day 0 and a positive test on Day 21, indicating pregnancy, calved, while 16% of the cows with a negative test on Day 0 and Day 21, indicating nonpregnancy, turned out to be pregnant. Of the 82 animals with P4 levels lower than 1 ng/ml on Day 0 and higher than 1 ng/ml on Day 21, only 61.0% calved. All 14 cows with low levels both on Day 0 and Day 21, indicating nonpregnancy, were found to be not pregnant. The influence of both early embryonic death and the accumulation of intrauterine fluids on the accuracy of these tests are discussed.

Peripheral plasma concentrations of oestradiol, progesterone, cortisol, LH and prolactin during the oestrous cycle in the cow, with emphasis on the peri-oestrous period

Abstract Interrelationships of circulating hormone levels and their implications for follicular development were studied throughout the oestrous cycle with emphasis on the perioestrous period in heifers and cows. The oestradiol level showed a major peak (45 pmol/1) before and coinciding with oestrus, and a second peak (27 pmol/1) around day 5–6 (day 0: day of first standing oestrus); it was low during the luteal phase of the cycle when progesterone was higher than 14 nmol/1 from day −12 to day −2. Large antral follicles, which had developed during the luteal phase, did not secrete significant amounts of oestradiol, degenerated after luteolysis, and were replaced by a newly developing follicle which became preovulatory. Parallel with this development the oestradiol level increased from the onset of luteolysis to reach a plateau about 26 h before the onset of oestrus. The interval between the onset of luteolysis and the onset of oestrus was 58 h; luteolysis proceeded at a slower rate in heifers than in cows. At 4.6 h after the onset of oestrus the maximum of the LH surge was recorded; the LH surge appeared to be postponed in the period October–December in comparison to the period August–September. The maximum of the LH surge was higher in heifers (45 μg/l) than in cows (30 μg/l), but its duration was similar (8.0 h). The oestradiol level decreased significantly from 6 h after the maximum of the LH surge, and standing oestrus (duration 18 h) was terminated almost at the same time as the return to basal values of oestradiol. Cortisol and prolactin levels did not show a peak during the peri-oestrus period. Cortisol fluctuated irrespective of the stage of the oestrus cycle and prolactin was significantly higher during the luteal phase. The results of this study indicate that development of the preovulatory follicle starts in the cow at the onset of luteolysis, about 2.5 days before the preovulatory LH surge, and that oestradiol secretion by this follicle is possibly inhibited by the LH surge.

Termination of mid-gestation pregnancy in bitches with aglepristone, a progesterone receptor antagonist

Six pregnancies were terminated in mid-gestation with aglépristone, a progesterone receptor antagonist, in 5 beagle bitches in order to determine the effects of aglépristone on plasma concentrations of prolactin and progesterone, the duration of the luteal phase, and the interestrous interval. In addition, the effects of aglépristone on the condition of the uterus and fetuses were examined by ultrasonography. After confirmation of pregnancy by ultrasonography, the dogs received 10 mg, s.c. aglépristone per kg body weight on 2 consecutive days at about 30 d post ovulation. Before, during and after treatment with aglépristone, plasma samples were collected for determination of the concentrations of prolactin and progesterone. The condition of the uterus and fetuses was assessed by ultrasonography the day before and at least 3 times a week for at least 2 wk after aglépristone administration. Termination of pregnancy occurred within 4 to 7 d after the start of aglépristone treatment, which was well tolerated, with no side-effects except slight vaginal discharge. The results of ultrasonographic examination indicated that aglépristone leads to abortion but not to fetal resorption. Elevated plasma concentrations of prolactin were observed during aglépristone treatment, while plasma progesterone levels remained unchanged. Pregnancy termination with aglépristone resulted in premature cessation of luteal function. In addition, the interestrous interval was shortened. The latter effects may be the consequence of actions of the progesterone receptor antagonist at the hypothalamus-pituitary level. In conclusion, aglépristone proved to be a safe and effective abortifacient in mid-gestation in the bitch. The results of the present study also indicated that aglépristone directly or indirectly influences pituitary function.