Cow Embryos Research Articles

160 EFFECT OF BSA SOURCES AND TYPES ON IN VITRO-PRODUCED LIVESTOCK EMBRYO DEVELOPMENT

Bovine serum albumin (BSA) is a macromolecule supplement used in embryo and cell culture media. Other chemicals have been used as macromolecule substitutes in embryo culture with variable effectiveness. There are BSA products available that are defined in their disease status, collection method, and manufacturing process. They are compliant for use in raw form or in culture medium in the USA and EU. It was the purpose of this study to compare the effectiveness of highly defined and internationally compliant BSA with typically used BSA on in vitro-produced pig and cow embryo development. Pig oocyte-cumulus complexes were matured in two stages for 44 h in vitro. Semen from one boar of known high fertility was used across the study to fertilize mature oocytes. After 6-h co-incubation with sperm (50 motile sperm/oocyte), presumptive zygotes were cultured for 120 h in modified NCSU-23 containing 4 mg/mL of one of the following heat-shocked BSA fractions: Sigma A-7906 (control), Minitube Reproductive Biology Grade Fraction-V (RBG-V1, RBG-V2, RBG-V3), or Minitube Reproductive Biology Grade Fatty Acid-Free (RBG-FAF1). Across all treatments, Day 5 morulae were removed from BSA culture and placed into modified NCSU-23 with 10% fetal bovine serum (FBS) (no BSA) and cultured for 48 h. After 168-h total culture, the following blastocyst development was observed: Sigma A-7906, 12.3% (40/324); RBG-V1, 21.1% (36/171); RBG-V2, 19.0% (30/158); RBG-V3, 16.8% (27/161); and RBG-FAF1, 13.4% (21/157). These data show that culture medium supplemented with Minitube Reproductive Biology Grade BSA meets or exceeds (P < 0.05; ANOVA-GLM of SAS; SAS Institute, Inc., Cary, NC, USA) blastocyst development potential when compared to culture medium supplemented with undefined standard BSA preparations, such as Sigma A-7906. In vitro-matured cow cumulus-oocyte complexes were fertilized and cultured in one of two CR-1aa-based media. Oocytes were fertilized in IVF medium containing 6 mg/mL Sigma A-8806 (FAF) or 6 mg/mL Minitube RBG-FAF1. After 24 h in IVF medium, presumptive zygotes from a specific BSA-supplemented medium were cultured for 144 h in CR-1aa supplemented with their respective BSA (8 mg/mL). Day 6 morulae were removed from BSA culture and placed into CR-1aa with 10% FBS (no BSA) and cultured for 48 h. After 192-h total culture, the following blastocyst development from oocytes matured was observed: Sigma A-8806, 21.4% (88/411); RBG-FAF1, 18.9% (70/370). These data show that culture medium supplemented with Minitube Reproductive Biology Grade BSA meets blastocyst development potential (P > 0.05) when compared to culture medium supplemented with undefined standard BSA preparations, such as Sigma A-8806. The inclusion of internationally compliant BSA meets or exceeds blastocyst development rates in comparison to standard BSA preparations in common in vitro embryo production systems for swine and cattle. Although manufacturing differences remain the prominent variant in BSA sources and types, continued monitoring and documentation of BSA preparations tested in livestock in vitro embryo production systems will ensure a safe global supply of BSA products for future culture media production.

333PREGNANCY RATE OBTAINED WITH EMBRYOS COLLECTED AFTER INSEMINATION OF SUPEROVULATED COWS WITH SEXED SORTED SEMEN

According to the production objectives of beef or dairy commercial farms, the possibility of gender selection of the offspring is an important tool to optimize the use of genetic and economical resources. The use of sexed sorted semen by flow cytometry to inseminate donors in a MOET program has successfully demonstrated a normal embryo production per cow (Theriogenology 59:513). The aim of this study was to evaluate the pregnancy rate and sex accuracy obtained after transferring embryos collected using sexed semen to inseminate the donors in MOET programs. The semen was provided by our commercial laboratory. Eighteen cows were inseminated with frozen female sexed semen and 22 with frozen male sexed semen. Embryos were collected using a commercial flushing medium with BSA and antibiotics. After the embryos were washed through 10 drops of holding medium, 50 embryos were transferred fresh;; 150 were frozen in 1.5M ethylene glycol, thawed 10 seconds in air;; and held for 30 seconds in a water bath at 35°C before transferring them. Fresh embryo stage and quality rates were: morula grade 1: 49%, grade 2: 13%; blastocyst grade 1: 34%, grade 2: 4%. Frozen embryo stage and quality rates were: morulae grade 1: 28%, grade 2: 18%; blastocyst grade 1: 47%, grade 2: 7%. The embryo transfers (ET) were performed in previously synchronized recipients injected with two doses of prostaglandin 12 days apart. Thirty days after ET, the diagnosis of pregnancy was done by transrectal ultrasonography (ALOKA 500 Scanner) to asses the pregnancy rate. Sixty days after the ET, fetal sex determination was done. The results are shown in the table 1 below. The pregnancy rates in both fresh and frozen-thawed preselected sex embryos are similar to those obtained with nonpreselected sex embryos (Theriogenology 56:1401). The high rate of sex accuracy shows the reliability of the technique of sexed sorted semen by flow cytometry to produce preselected sex embryos in superovulated cows. This research was supported by Fundación Margarita Perez Companc. Table 1

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) expression and activity during early embryo development in the cow.

Several extracellular matrix (ECM)-degrading proteinases are hypothesised to play important roles during early mammalian development. In particular, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are expressed in peri-implantation mouse, sheep, and pig embryos and are implicated in the implantation process. These proteinases are not expressed in early (pre-blastocyst) mouse, sheep or pig embryos. The aim of this study was to establish the gene expression and proteolytic activity of uPA and MMP-9 in in vitro-produced (IVP) cow embryos. Using RT-PCR, mRNA transcripts for uPA and MMP-9 were detected during the first 7 days of development. To investigate the activity of these proteinases, conditioned media from various stages of development (days 2, 3, 4, 5 and 7) were assayed for uPA activity by chromogenic assay and MMP-9 activity by gelatin zymography. Both uPA and MMP-9 activities were detected in the media samples indicating the production and secretion of these proteinases. This pattern of proteinase expression is novel in comparison to the mouse where uPA and MMP-9 are only expressed from the blastocyst stage onwards. The results of this study suggest that these ECM proteinases have a role prior to implantation in the cow, in contrast to that exhibited by mouse, sheep and pig embryos.

Transfer of Beef Embryos in Dairy Cows: Influence of Recipient and Embryo Quality on Pregnancy Rate and Calving Performance

ContentsOn three experimental farms 205 deep frozen Blonde d’Aquitaine embryos were transferred into Holstein dairy cows 7 days after a spontaneous heat. The aim of the study was firstly to examine the influence of recipient and embryo quality on pregnancy rate and secondly to study the calving performance of dairy cows carrying beef embryos. Recipients were divided into three groups based on the quality of the corpus luteum (unsuitable, moderately suitable and suitable) established by rectal palpation. Embryos classified as fair, good and excellent, were randomly distributed over the recipients, including those classified as being unsuitable. The overall pregnancy rate was 36% (standard deviation (SD) = 3.3), dystocia occurred in 48% of births (SD = 5.9), gestation was 297 days (SD = 1.2) and birth weight was 52 kg (SD = 2.0). No statistical differences were found in pregnancy rates and calving performance between the groups of recipients or between the classes of embryos. It is concluded that there is no justification to reject recipients on the basis of the quality of the corpus luteum. Furthermore there is no reason to reject fair embryos for transfer. Beef embryos in dairy cows resulted in much dystocia because of high birth weight, and in long gestation periods.

Transfer of Beef Embryos in Dairy Cows: Influence of Recipient and Embryo Quality on Pregnancy Rate and Calving Performance

Transfer of Beef Embryos in Dairy Cows: Influence of Recipient and Embryo Quality on Pregnancy Rate and Calving Performance

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.

The fear of cloning

The fear of cloning

Taxa de gestação e morfologia de embriões bovinos da raça Nelore resfriados por 24 horas a 5°C em contêiner modelo Celle modificado

Na tentativa de se estabelecerem condições de estocagem de embriões bovinos da raça Nelore, resfriaram-se 29 embriões em um contêiner modelo Celle modificado, por 24 horas a 5°C. Os embriões foram avaliados, classificados quanto ao estádio de desenvolvimento e qualidade embrionária, antes e após o resfriamento, e transferidos a receptoras sincronizadas à base de prostaglandina e progestágeno. Dos embriões resfriados, 82,8% (24/29) eram grau I, 13,6% (3/29) grau II e 6,9% (2/29) grau III. Após 24 horas de resfriamento, 62,1% (18/29) dos embriões continuaram grau I, 20,7% (6/29) grau II e 17,2% (5/29) grau III. Durante o resfriamento, todos os embriões permaneceram no mesmo estádio de desenvolvimento. Porém, dos embriões na fase de blastocisto inicial a blastocisto expandido, 82,8% (24/29) apresentaram colapso da blastocele. Após a transferência, obtiveram-se 37,9% (11/29) de gestação positiva, inferior (P<0,05) àquela apresentada pelo grupo-controle, de 75,6% (62/82). Possivelmente, o método de sincronização influenciou os resultados, obtendo-se apenas uma gestação positiva das 11 receptoras que receberam progestágenos.

Birthweighits of calves conceived by transfer of Japanese black cow embryos produced in vitro by co-culture with cumulus cells

To study the effect of human follicular fluid (FF) and the specific contribution of its epidermal growth factor (EGF) component on the in vitro maturation of cumulus-enclosed mouse oocytes.A previously described mouse oocyte model system was used to study the effect of FF on oocyte maturation before and after extraction of EGF by immunoprecipitation. Follicular fluid specimens enclosing both mature and immature human oocytes were tested.The endpoints assessed were the percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation at different intervals over a 24-hour period and the final degree of cumulus expansion achieved.A concentration-related stimulatory effect of mature FF was noted when compared with the spontaneous increase of GVBD and polar body one formation observed for the EGF-free control medium. Overall, the effect of immature FF was inhibitory. After extraction of EGF from FF by immunoprecipitation from both immature and mature FF, the rates of GVBD and polar body one formation were decreased in both groups. The addition of 5 ng/mL of EGF to the extracted groups reversed this effect on polar body one formation. Cumulus expansion was maximal for oocytes incubated with mature FF and minimal for those incubated with EGF-free media.The positive effect of mature human FF on mouse oocyte maturation and cumulus expansion is to a large extent because of the presence of EGF.

The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer

We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow= 138±46 vs calf= 142±59; mean±SD) or ICM and TE cell number between calf (ICM= 35 ± 19, TE= 107± 52) and cow (ICM= 38± 21, TE= 99 ± 32) blastocysts, nor in the ICM/total cell number ratio (cow= 0.27± 11, calf= 0.25 ± 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, althrough this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identifed between these 2 groups of embryos.

Media and time of oocytes transport influence their developmental competence for in vitro production of bovine embryos

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20–22°C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4°C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22°C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20–22°C, the embryo survival rate decreased (PBS + albumin) or no embryo survived (TCM199 + calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.

Adenovirus-mediated gene transfer by perivitelline microinjection of mouse, rat, and cow embryos.

To determine the fate of an episomally expressed transgene, mouse, rat, and cow zygotes were injected into the perivitelline space with approximately 100 pl of buffer containing the replication-defective human adenovirus, AdCMVLacZ/sub360. Viral concentrations ranged from 2.5 to 2.5 x 10(5) plaque-forming units (pfu)/100 pl. As viral titer increased, fewer embryos were able to develop to blastocysts. In the mouse, the percentage of blastocysts formed ranged from 82% in controls to 16% after injection at the highest titer. In the rat and cow, a similar decrease in blastocyst formation was noted (62% to 6% and 26% to 4%, respectively). Reporter gene (galactosidase, LacZ) activity could be detected in mouse embryos after injection at a concentration of only 25 pfu/100 pl, whereas a tenfold higher titer was required in the other two species to observe the blue LacZ reaction product. When examined after 5 (mouse), 6 (rat), or 9 (cow) days of in vitro culture, the proportion of LacZ-positive embryos ranged from 15% to 96%, 6% to 76%, and 18% to 58% in mouse, rat, and cow embryos, respectively, depending upon viral concentration. However, a large percentage of positive embryos proved to be expression mosaics, the degree of which was likewise dependent on titer. While none of the embryos showed LacZ activity at 30 h after injection, 70% of mouse, 8% of rat, and 20% of cow embryos expressed the reporter gene at 42 h. Delaying the timing of injection revealed that the efficiency with which mouse and rat embryos could be infected decreased with increasing degree of differentiation. Only 35% and 18% of mouse embryos expressed the reporter gene after injection at the morula or blastocyst stage, respectively. A similar drop in efficiency was noted in rat embryos when injections took place at the 8-cell, morula, or blastocyst stage, with 70%, 33%, and 9% of embryos, respectively, subsequently showing LacZ activity. Likewise, advanced development resulted in a decrease in the efficiency of viral-mediated gene transfer in cow embryos, with 100%, 78%, and 68% of embryos being positive after injection at the 8-cell, morula, or blastocyst stage, respectively. These results demonstrate that a human adenovirus can be used to express a reporter gene transiently in nonhuman embryos.

Birthweights of calves conceived by transfer of Japanese black cow embryos produced in vitro

Birthweights of calves conceived by transfer of Japanese black cow embryos produced in vitro

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration.

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zone dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS+albumin, TCM199 and TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.

Glucose controls sex-related growth rate differences of bovine embryos produced in vitro

The objective of this study was to determine whether the presence of glucose in the culture medium causes more growth of male embryos than of female embryos in cows. Oocytes matured and fertilized in vitro were cultured from 24 to 48 h after insemination in CR1aa medium with or without 5.56 mmol glucose l-1. At the end of culture the mean numbers of cells of the cleaved embryos were 5.07 (+/- 0.16) in glucose-free medium and 5.29 (+/- 0.17) in medium with glucose (P = 0.86). Without glucose in the medium, the least square mean was 0.22 cells higher in males than in females. This difference was not significant (P = 0.44). However, in the presence of glucose this difference was 1.25 cells in favour of males (P = 0.0001). The possibility that the sex difference is linked with growth stimulating effects of oxygen radicals is discussed.

Sex of IVM, IVF and IVC Blastocysts Produced from Individual Donor Cows.

This experiment was undertaken to determine if developmental rate to the blastocyst stage and the blastocyst production capacity of donor cows affects embryo sex ratio, and to clarify the relationship between individual donor cows and male sex predominance of embryos that reach blastocyst stage relatively early. In vitro matured (IVM), in vitro fertilized (IVF) and in vitro cultured (IVC) embryos produced separately from donor cows were used. The sex of the embryos at the blastocyst stage was determined by PCR analysis. Although there was no significant difference in the sex ratio of embryos in relation to days after insemination, a higher proportion of male blastocysts was obtained on days 6.5 and 7.0 (59.3% and 58.0%, respectively) than on days 7.5 and 8.0 (50.7% and 49.3%, respectively). No relationship between male predominance and the developmental rate of embryos was found. Nor was there any evidence of a characteristic relationship between the developmental rate and the sex of embryos produced from individual donor cows. However, when donor cows were classified according to the period required for their embryos to reach blastocyst stage, a higher percentage of cows showed a higher male percentage in their embryos on the least number of days after insemination than on later days. This difference also was not significant. Therefore, there is little relationship between embryonic sex and the developmental rate of embryos in cows.

Expression of Genes Encoding Antioxidant Enzymes in Preimplantation Mouse and Cow Embryos and Primary Bovine Oviduct Cultures Employed for Embryo Coculture1

Preimplantation embryos from a variety of mammalian species contrast markedly in their response to culture in vitro. Murine preimplantation embryos display a wider tolerance than other mammalian species to culture environments, and this has contributed to the development of several effective defined culture media. Embryo coculture on somatic cells remains the most effective method of supporting reasonable rates of bovine preimplantation development in vitro. The patterns of gene expression for several antioxidant enzymes during preimplantation murine and bovine development were examined by use of the reverse transcription-polymerase chain reaction technique to determine whether the differential developmental capacity of mammalian preimplantation embryos in culture may reflect variations in the patterns of expression for a series of antioxidant enzymes. Transcripts for catalase, CuZn-containing superoxide dismutase (CuZn-SOD), Mn-SOD, glutathione peroxidase (GPX), and glutamylcysteine synthetase (GCS) were detected in mouse embryos at all stages of development regardless of in vivo or in vitro development. Preimplantation cow embryos produced by in vitro procedures expressed mRNAs for catalase, CuZn-SOD and GPX, whereas transcripts for Mn-SOD were not detected at any stage. GCS transcripts, although present in stages up to the morula, were not detected in cow blastocysts. Analysis of antioxidant gene expression in both bovine primary oviductal cell monolayer cultures and nonattached, ciliated oviductal cell vesicle cultures revealed a constitutive pattern of expression of all five enzymes for the 8-day culture interval. These experiments suggest that differences in gene expression may contribute to the variation in the ability of embryos to develop in vitro with respect to levels of oxygen and dependence on coculture.

Binding of epidermal growth factor and transforming growth factor-α in mammalian preimplantation embryos

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.

Developmental Changes in Embryonic Resistance to Adverse Effects of Maternal Heat Stress in Cows

The objective of this study was to determine whether bovine embryos become more resistant to deleterious effects of maternal heat stress as early embryonic development progresses. Superovulated, lactating Holstein cows were bred by AI and assigned to be heat stressed on d 1, 3, 5, or 7 of pregnancy (d 0 = day of estrus) or not heat stressed (control). Embryos were retrieved from the uterus on d 8 and evaluated for viability and stage of development. Compared with embryos of control cows, embryos of cows receiving heat stress on d 1 had decreased viability and development. Maternal heat stress on other days had no detrimental effect on embryonic viability or stage of development. Bovine embryos become more resistant to adverse effects of maternal heat stress as pregnancy progresses; substantial resistance develops by d 3. This information may be useful in design of environmental modification systems that provide cooling at critical periods of gestation to enhance pregnancy rates during summer in hot climates.

Expression of IGF ligand and receptor genes during preimplantation mammalian development.

The temporal patterns of expression of genes encoding insulin-like growth factor (IGF) ligands and receptors during very early development have been investigated in several laboratories in several different mammalian species. Both reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical techniques have been used to identify the time of appearance of gene transcripts or end-products. In preimplantation mouse embryos, IGF-II ligand and receptor gene activity is detectable as early as at the two-cell stage, the time when transcription from the embryonic genome is activated, but receptors for insulin and IGF-I are not detectable until the compacted eight-cell stage. Transcripts for insulin or IGF-I are not detectable in preimplantation mouse embryos, although the ligands are present in the reproductive tract. The pattern of IGF gene expression is not, however, identical in all mammalian species. In cow embryos, for example, transcripts for IGF-I and IGF-II ligands and receptors and insulin receptors have been detected at all stages of preimplantation development from mature oocyte to blastocyst (Watson et al., 1992). Attempts to quantitate transcript abundance in these early embryos are in progress in our laboratory. In the preimplantation mouse embryo, transcripts for several different IGF-binding proteins (IGFBP-2, -3, -4, and -6) have been detected by RT-PCR procedures. In addition, transcripts for IGFBPs have been identified in RNA derived from cumulus cells, the ovary, the oviduct, the uterus, and the decidua. These findings suggest that the interactions of IGF ligands and receptors in preimplantation development might, indeed, be modulated by IGFPs.(ABSTRACT TRUNCATED AT 250 WORDS)