Notochord isolation using laser capture microdissection.

BACKGROUND: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. METHODS: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. RESULTS: This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. CONCLUSION: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

Morphology of the human cervical vagus nerve: implications for vagus nerve stimulation treatment.

OBJECTIVES: The vagus nerve has gained a role in the treatment of certain diseases by the use of vagus nerve stimulation (VNS). This study provides detailed morphological information regarding the human cervical vagus nerve at the level of electrode implant. RESULTS: Eleven pairs of cervical vagus nerves and four pairs of intracranial vagus nerves were analysed by the use of computer software. It was found that the right cervical vagus nerve has an 1.5 times larger effective surface area on average than the left nerve [1,089,492 ± 98,337 vs 753,915 ± 102,490 µm(2), respectively, (P < 0.05)] and that there is broad spreading within the individual nerves. At the right side, the mean effective surface area at the cervical level (1,089,492 ± 98,337 µm(2)) is larger than at the level inside the skull base (630,921 ± 105,422) (P < 0.05). This could imply that the vagus nerve receives anastomosing and 'hitchhiking' branches from areas other than the brainstem. Furthermore, abundant tyrosine hydroxylase (TH)- and dopamine ß-hydroxylase (DBH)-positive staining nerve fibres could be identified, indicating catecholaminergic neurotransmission. In two of the 22 cervical nerves, ganglion cells were found that also stained positive for TH and DBH. Stimulating the vagus nerve may therefore induce the release of dopamine and noradrenaline. A sympathetic activation could therefore be part of mechanism of action of VNS. Furthermore, it was shown that the right cervical vagus nerve contains on average two times more TH-positive nerve fibres than the left nerve (P < 0.05), a fact that could be of interest upon choosing stimulation side. We also suggest that the amount of epineurial tissue could be an important variable for determining individual effectiveness of VNS, because the absolute amount of epineurial tissue is widely spread between the individual nerves (ranging from 2,090,000 to 11,683,000 µm(2)). CONCLUSIONS: We conclude by stating that one has to look at the vagus nerve as a morphological entity of the peripheral autonomic nervous system, a composite of different fibres and (anastomosing and hitchhiking) branches of different origin with different neurotransmitters, which can act both parasympathetic and sympathetic. Electrically stimulating the vagus nerve therefore is not the same as elevating the 'physiological parasympathetic tone', but may also implement catecholaminergic (sympathetic) effects.

Expression of progesterone receptor related to the polymorphism in the PGR gene in the rabbit reproductive tract.

The association of the 2464G > A SNP found in the promoter region of the rabbit progesterone receptor gene with progesterone receptor (PR) expression was evaluated by Western blot analysis. This SNP was associated with 2 lines divergently selected for uterine capacity, the high line selected to increase uterine capacity and the low line selected to decrease uterine capacity. Two progesterone isoforms were obtained using a commercial monoclonal antibody: the PR-B isoform described previously in rabbits, and the PR-A isoform, not described previously in rabbits. The GG genotype, the genotype more frequent in the high line, showed less PR-B and PR-A expression than the AA genotype in the oviduct (GG/AA(PR-B) = 0.81 and GG/AA(PR-A) = 0.73) and uterus (around 0.70 in both isoforms). The GA genotype showed similar PR-A expression in both tissues and also similar PR-B expression in the oviduct to the GG genotype. Conversely, the GG genotype showed less PR-B expression than the GA genotype in the uterus (GG/GA(PR-B) = 0.79). Similar expression of both PR isoforms was found in the uterus at d 2 and 3 of gestation; meanwhile, an increase of both isoforms was observed in the oviduct. Similar PR-A expression was observed in the ampulla and isthmus; meanwhile, the PR-B expression in the isthmus was double that in the ampulla.

Is the zona pellucida an efficient barrier to viral infection?

Although the transfer of embryos is much less likely to result in disease transmission than the transport of live animals, the sanitary risks associated with embryo transfer continue to be the subject of both scientific investigations and adaptations of national and international legislation. Therefore, the implications are important for veterinary practitioners and livestock breeders. In vivo-derived and in vitro-produced embryos are widely used in cattle and embryos from other species, such as sheep, goats, pigs and horses, are also currently being transferred in fairly significant numbers. Bearing in mind the wide variety of embryos of different species and the correspondingly large number of viruses that are of concern, it is expedient at this time to look again at the importance of the zona pellucida (ZP) as a barrier against viruses and at the susceptibility or otherwise of embryonic cells to viral infection if ever they are exposed. For embryos with an intact ZP, viral infection of the embryo is unlikely to occur. However, the virus may stick to the ZP and, in this case, International Embryo Transfer Society (IETS) washing procedures in combination with trypsin treatment are mandatory. A caveat is the fact that currently more and more types of embryos are becoming available for transfer and scientific data cannot be extrapolated from one species to another. These topics are discussed in the present review.

Haptoglobin expression and release by rabbit oviduct and endometrium, its localization in blastocyst extra-embryonic matrix and fluid during preimplantation time.

BACKGROUND: Evidence is emerging that haptoglobin, an acute phase protein with immunomodulatory properties, is expressed by the endometrium of various species. The present study describes an in-depth investigation of haptoglobin expression and release in the rabbit reproductive tract and in preimplantation embryos. METHODS: The full-length cDNA sequence of rabbit haptoglobin was determined by rapid amplification of cDNA ends PCR. Haptoglobin expression was studied in the oviductal ampull, and isthmus, endometrium and embryos from the time of ovulation up to adhesion. These results were completed by western blot analysis of reproductive tract secretions and embryonic tissues. RESULTS: cDNA sequencing showed a high homology between rabbit and human haptoglobin (84.1%). In oviductal tissues haptoglobin mRNA is clearly expressed from 6 h post-conception (p.c.) to day 3, and in the uterus on days 5 and 6. In the oviductal fluid highest haptoglobin protein content was found between 6 h p.c and day 2, and in the uterine fluid on days 5 and 6 p.c. Embryos do not express haptoglobin mRNA during preimplantation development. However, considerable amounts of maternal haptoglobin protein were detected in the blastocyst coverings and in blastocyst fluid. CONCLUSIONS: Already during periovulatory time and oviductal passage, high amounts of haptoglobin are present in the microenvironment surrounding the oocyte/embryo. Two days before implantation, again, high haptoglobin levels are detectable in the embryo's environment. The incorporation of haptoglobin into the extra-embryonic matrix may be of particular functional significance.

Embryo-maternal communication in bovine - strategies for deciphering a complex cross-talk.

Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.

Proteins in the extraembryonic matrix of preimplantation rabbit embryos.

Preimplantation embryos of several species are surrounded by an extraembryonic matrix (often simply named zona pellucida) until briefly before implantation. All signals of the early embryo-maternal dialogue have to pass this matrix and therefore are detectable inside. We investigated the protein pattern of the extraembryonic matrices of 3-6-day-old rabbit embryos by two-dimensional gel-electrophoresis. Using (35)S-methionine incorporation, embryonic proteins were labelled and could be distinguished from maternal proteins. Furthermore, the presence of three different proteins (insulin-like growth factor-binding protein-3, uteroglobin, haptoglobin) within the matrices of day-6 embryos was investigated by Western blot analysis. The pattern and numbers of protein spots detected was clearly dependent on the time of embryonic development. Of all proteins detected, 19.3% and 33% are of embryonic origin (day 5 and day 6, respectively). At day 4 the zona proteins are no longer detectable, reflecting the degradation of the zona pellucida. From day 4 to day 5 proteins detectable within the extraembryonic matrices increase enormously. This demonstrates that embryo-maternal signalling accelerates at least 2 days before implantation. Insulin-like growth factor-binding protein-3, uteroglobin and haptoglobin are part of the early signalling as shown by Western blot analysis. Insulin-like growth factor-binding protein-3 could be detected as one spot at 38 kDa pI 6.1, uteroglobin at 8 kDa pI 6.0 and haptoglobin as two spots/isoforms at 36/38 kDa pI 5.8 and pI 6.0. These results demonstrate that extraembryonic matrices serving as a mailbox are a valuable tool for investigating early embryo-maternal signalling.

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits.

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.

Modulation of endometrial transformation in gonadotrophin-stimulated and unstimulated pseudo-pregnant rabbits: studies with the progesterone receptor antagonist, onapristone.

Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.

Horse conceptuses secrete insulin-like growth factor-binding protein 3.

Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radiolabeled proteins showed that cultured horse conceptuses secreted IGFBP3 into the culture medium. Endometrial samples from mares also contained IGFBP3 mRNA and protein; but there was no evidence of secretion of IGFBP3 into the uterine lumen by ligand blot analysis, and there was evidence of only very small amounts by Western blot analysis. These results indicate that the horse conceptus secretes significant quantities of IGFBP3 toward the conceptus capsule from as early as Day 10 after ovulation. Thus, most of the IGFBP3 contained within the capsule, which binds IGF-I to this special extracellular matrix of the preimplantation horse conceptus, is likely to be embryonic in origin. IGFBP3 in the horse conceptus capsule may enhance or modulate the action of IGFs on the developing conceptus.

Early embryonic coats: morphology, function, practical applications. An overview.

Mammalian egg and embryo coats are primarily represented by the zona pellucida which, however, undergoes biochemical and structural changes during fertilization and embryo development. It serves several functions, from ovulation until shortly before implantation. Initially the zona pellucida induces sperm-oocyte interaction, acrosome reaction and prevents polyspermy. Later, it prevents disaggregation of the noncompacted blastomeres and the premature attachment to the oviductal and endometrial surface. Additionally, it protects the embryo against toxins and xenobiotics, as well as bacteria, viruses and phagocytes. As the embryo is covered by the zona pellucida until immediately before implantation, all signals of embryo-maternal signalling have to pass the zona and are detectable within it. Logically we may define the zona pellucida as a mailbox of the embryo-maternal signalling, especial for investigating these messages. Oviductal, uterine and embryonic proteins are incorporated into the zona pellucida as embryonic development goes on and change the zona's morphological and biochemical properties. Whether a protein is able to penetrate the zona, whether it accumulates within the zona or whether it is rejected by the zona depends on its biochemical properties. Three specific proteins have been detected within the embryonic coats (IGFBP3, HB-EGF, P19). New insights into the physiology of the zona pellucida might present new achievements in the in vitro culture of embryos, and present new aspects as to how to prevent zona hardening. Furthermore, knowledge of the zona proteins enables the development of immunocontraceptive vaccines. Consequently, the zona pellucida is not only significant with regard to fertilization, but also during early embryonic development. Investigations of the zona pellucida will yield new insights into early embryo-maternal signalling which in turn may lead to improvements in classic IVF and new approaches to in vitro culture.

Identification of proteins in the equine embryonic capsule.

An acellular embryonic capsule envelops equine conceptuses between day 6 and day 23 after ovulation. As all of the factors mediating embryo-mother signalling must pass through the capsule, it acts like a 'mailbox'. Therefore, we have started to map the proteins in this special extracellular matrix at the interface between mother and embryo. In the present study, one- and two-dimensional gel electrophoresis were used to examine a range of proteins. Use of western blotting identified three specific proteins in the capsules of equine conceptuses recovered on day 16 after ovulation: insulin-like growth factor binding protein 3 (IGFBP-3), a 19 kDa uterine lipocalin (P19) and heparin-binding epidermal growth factor (HB-EGF). Western blotting of two-dimensional SDS-PAGE gels revealed the isoelectric points (pI values) of these proteins: IGFBP-3 was detected as the non-glycosylated 32 kDa form with two isoforms at about pI value 5.8; P19 had a pI value of 9.1; and several isoforms of HB-EGF were detected with molecular masses of approximately 28 kDa and a pI value range of 5.8-6.2. The origin of HB-EGF is not known, but IGFBP-3 is embryonic and P19 is maternal in origin and is thought to be a transport protein. In addition to playing a protective role, and probably also contributing to the mobility of the young conceptus within the uterus, the capsule may be thought of as the extracellular matrix of the embryo, which modulates the complex embryo-maternal signalling processes that take place during early pregnancy in mares.

Flow cytometry to monitor phagocytosis and oxidative burst of anaerobic periodontopathogenic bacteria by human polymorphonuclear leukocytes.

The reduced susceptibility to phagocytosis found among some periodontopathogenic anaerobes may account for the differences between invasive and non-invasive strains. We applied flow cytometry as a powerful tool to analyze and quantify phagocytosis using standardized cultures of oral anaerobes (Porphyromonas gingivalis, Prevotella intermedia, P. nigrescens, Capnocytophaga gingivalis, C. ochracea, C. sputigena, Fusobacterium nucleatum and Peptostreptococcus micros) and heparinized whole blood. Bacteria were labeled by a fluorescein-methylester and their esterase activity, resulting in green fluorescence. Ingested bacteria could be detected easily and quantified by a shift towards green fluorescence in the PMNL population involved and a concomitant decrease in the bacterial population. Furthermore, the oxidative burst of PMNLs was detected in parallel assays using the dye DHR123 which becomes fluorescent upon oxidation during the oxidative burst process. We found a great diversity in phagocytosis susceptibility determined by estimating the portion of phagocytosing PMNLs, ranging from 10.6% (strain W83) to > 99.4% (e.g. ATCC 33277T) in P. gingivalis and from 15.9% (strain MH5) to > 95% (ATCC 33563T) in P. nigrescens. In contrast, almost all P. intermedia strains as well as the representatives of the other anaerobic, putative periodontopathic species tested showed no or only moderate resistance in the phagocytosis assay. Comparison of clinical data of patients and the extent of phagocytosis resistance of the corresponding P. gingivalis strains suggests that this virulence factor may contribute to the clinical outcome.

Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis.

Insulin as well as insulin-like growth factor-I (IGF-I) promote early embryo development, and IGF-I binds to the coats of preimplantation rabbit embryos. As the IGF-I receptor is expressed from the morula stage onwards, the embryos are capable of responding to insulin and IGF-I, which is present in the oviductal and uterine secretions that surround them. The embryonic coats were removed to exclude any influence by IGF-I bound to the coats. The in vitro development of such embryos under classical conditions appears to be retarded. Addition of IGF-I (68 pM-6.8 nM) or insulin (68 nM-6.8 microM), however, promotes blastocyst formation. Embryo development under such conditions is not significantly different from that of embryos cultured with intact coats. In contrast, coat-free embryos cultured without IGF-I or insulin supplementation show apoptosis. Because IGF-I stimulates cell proliferation and prevents apoptosis, we investigated whether insulin or IGF-I may act as "survival factors" in preimplantation development. Therefore, apoptosis was induced by slight UV irradiation (254 nm wave length; 11.8 W/m2). Compared to the untreated controls, embryos displaying retarded development or degeneration were increased by 22% and 14%, respectively. Addition of IGF-I or insulin to the culture medium of UV-irradiated embryos improved [3H]thymidine incorporation and blastocyst formation significantly. By immunohistochemistry we could show that addition of insulin (0.68-68 nM) decreased apoptosis and increased cell proliferation in a dose-dependent manner, supporting blastocyst development significantly.

Transfer of a uterine lipocalin from the endometrium of the mare to the developing equine conceptus.

One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived P19 fusion protein. The antiserum raised against the C-terminal peptide recognized P19 on Western blots of denatured uterine secretions (subjected to SDS-PAGE), but it did not bind to the protein in tissue sections. However, the antiserum raised against the recombinant-derived fusion protein recognized P19 both on Western blots and in histological sections. Western blot analysis of tissues and fluids collected from early-pregnant mares demonstrated significant quantities of P19 in the endometrium and uterine secretions and in the embryonic capsule, the chorion, and the yolk sac fluid, showing that the protein is transferred through to the developing embryo. Concentrations of immunoreactive P19 declined during gestation so that, by Day 30, it had virtually disappeared from both maternal and fetal tissues and fluids. Immunohistochemical staining of endometrial biopsies collected during early pregnancy localized P19 to the glandular and luminal epithelia and to the lumina of the endometrial glands. The capsule and the trophoblast layer of the chorion from early (Days 16-17) horse conceptuses also stained positively with localization of P19 to the apical surface of the trophoblast cells. There was no detectable staining either in or on the embryonic disc. The presence of P19 in both the trophoblast layer and the yolk sac fluid suggests that P19 passes into the yolk sac fluid via trophoblast cells.

Binding of IGF-I to preimplantation rabbit embryos and their coats.

It is known that insulin-like growth-factor I (IGF-I) promotes early embryonic development from the morula to the blastocyst stage in rabbits (28). Therefore we used autoradiography to investigate whether IGF-I binds to preimplantation embryos and its coats. From Day 3 after mating onwards, a clear binding of IGF-I to the embryos was observed. There was no difference in binding to the embryoblast or trophoblast cells. Using ligand blot, several IGF-binding proteins (IGFBP; 31 kDa, 33 kDa, 36 kDa, three overlapping bands at 40 to 55 kDa) were obvious in the embryoblast and trophoblast. A 120 to 130 kDa protein was observed exclusively in the embryoblast. Significant binding of (125)I IGF-I to the coats of embryos older than 3 d was detected, and IGF-I was bound via a 38 kDa protein, as detected by ligand blot. To investigate the origin of this protein, the patterns of IGFBP were determined in the oviductal and uterine fluids of pregnant animals (Days 0 to 6). The following binding proteins were observed regularly in the oviductal and uterine flushings: 28 kDa, 32 kDa and 3 overlapping bands in the area of 40 to 55 kDa. In the oviduct the main IGF binding protein was the 32 kDa band (38.7% to 45.9%), while in the uterus it was the 3 overlapping bands at 40 to 55 kDa (42.5% to 24.1%). Because IGF-I is produced in the oviduct and uterus (27), IGFBPs are found in oviductal and uterine fluids, IGF-I is stored in the coats, IGF-I binds to preimplantation embryos and IGF-I promotes early embryonic development (28), the IGF system seams to have a function in the maternal-embryonic interaction.

Superovulation of dairy cows with purified FSH supplemented with defined amounts of LH.

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<0.05) more corpora lutea (CL; 12.6+/-1.1) and fertilized ova (5.1+/-1.3) compared with Groups 2 and 4 (10.1+/-0.9 and 2.6+/-0.6, 9.0+/-0.9 and 2.7+/-0.5, respectively). Due to a high percentage of degenerated embryos (33%) Group 3 yielded only one more transferable embryo than Groups 2 and 4. Among groups, LH levels differed in the period prior to induction of luteolysis and were similar thereafter. The progesterone pattern following FSH/LH administration reflected the amount of LH supplementation. Milk progesterone levels on the day prior to embryo collection were correlated to the number of CLs and recovered embryos. It is concluded that under the conditions of our experiment superovulation with 0.423 IU LH/40 mg AU FSH may yield a significantly improved superovulatory response in dairy cows. It is further suggested that LH supplementation exerts its effects mainly on follicular and oocyte maturation during the period prior to luteolysis.

Rapid milk progesterone assay as a tool for the selection of potential donor cows prior to superovulation.

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<0.05) among groups, except in those with lower P(4) concentrations (1.1 +/- 0.0; 1.0 +/- 0.0; 3.7 +/- 1.5; 10.7 +/- 1.3; 13.8 +/- 1.3; 19.0 +/- 1.5 ng/ml, respectively). The correlation between RMPA-groups and EIA P(4) levels was 0.69 (P<0.001). Donors classified as having less P(4) than the standard yielded fewer corpora lutea (CL) (P<0.005), ova and embryos (P<0.05), and transferable embryos (P<0.05) compared with donors having similar or higher P(4) levels (3.4 +/- 1.0 vs 10.8 +/- 0.7 CL; 1.7 +/- 0.8 vs 6.2 +/- 0.9 ova and embryos; 1.2 +/- 0.7 vs 2.8 +/- 0.4 transferable embryos). Our results indicate that RMPA determines milk P(4) levels with sufficient accuracy and is a simple and useful tool for the screening of potential donor cows.