Higher air temperature in summer causes a significant reduction in fertility in cattle. Increase in female body temperature during the period of reproduction by only 2EC, also known as hyperthermia, leads to disturbances in the functioning of the female reproductive system, oocytes maturation, fertilization and embryos development. Particularly sensitive to high temperatures are embryos in the first and second day after fertilization (thermosensitive), but just at third till fifth day after fertilization their resistance to thermal stress significantly increases. Morula-stage and blastocyst-stage bovine embryos are insensitive to elevated temperatures (thermoresistant). Most probably this is due to the increasing number of cells within the embryo and the capacity to activate defense mechanisms based on the synthesis of various factors providing resistance to high temperatures. These factors include heat shock protein 70 (HSP70), antioxidants such as glutathione, and IGF-1. One of the responses of the embryo to elevated temperature is the induction of apoptosis, which is associated with the activation of embryonic genome. Owing to the apoptosis, cells damaged by high temperature may be eliminated from the embryo, which increases their chance of survival. Precise examination of the mechanisms responsible for the development of thermotolerance of preimplantation bovine embryos will enable their protection from the consequences of elevated temperature. The aim of this review is to summarise experiments in which in vitro embryo production system was used to estimate the influence of elevated temperature on cattle fertility.

This paper reviews the basic knowledge about obtaining farm animal embryos in vitro with special focus on differences among species and application of this procedure in the future. In vitro production of farm animal embryos consists of in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, and in vitro culture (IVC) of embryos. Oocytes can be collected from live animals (by laparotomy, laparoscopy, Ovum Pick Up) or from slaughtered ones (by puncture, sectioning). Usually immature oocytes are isolated, and during IVM they reach maturity. Matured oocytes are cultured with sperm (IVF), leading to the formation of zygotes. In the case of fertilization problems (horse, pig), intracytoplasmic sperm injection is used. The zygotes are usually cultured (IVC) to the morula and blastocyst stages. These embryos can be transferred to recipients or frozen/vitrified. Offspring have been obtained after transfer of cattle, sheep, goat, pig and horse embryos. This procedure can be used in animal breeding, biotechnology, medicine, and basic research.

The aim of this study was to evaluate the influence of elevated temperature on bovine oviduct epithelial cells (BOECs), based on the expression and localization of both heat shock protein 70 (HSP70), responsible for the cellular defence mechanism, and oviduct specific glycoprotein 1 (OVGP1) which is the most important embryotrophic protein. BOECs were cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated temperature (41°C) for 168 h. The elevated temperature had no effect on the viability of BOECs but exerted a negative effect on embryo development. The elevated temperature increased the expression of HSP70 and decreased the expression of OVGP1 at both mRNA and protein levels in BOECs cultured alone and those co-cultured with embryos. However, the presence of embryos limited the decrease in OVGP1 expression in BOECs at elevated temperature but did not alter the expression of HSP70. These results demonstrate for the first time the influence of elevated temperature on BOECs, consequently providing insights into the interactions between the embryo and the oviduct at elevated temperature.

Wisent (Bison bonasus), also called the European bison, is listed as vulnerable on the International Union for the Conservation of Nature Red List of Threatened Species. In Poland, a program for protection in situ and ex situ is being implemented. One new approach is the use of the in vitro embryo production (IVP) procedures to obtain wisent offspring. In contrast to previous successes with cattle IVP, use of IVP with wisent is limited by the small size of the population (only ~5000 individuals in more than 200 herds in Europe) and seasonal reproduction. The aim of this preliminary study was to obtain hybrid embryos (Bison bonasus × Bos taurus) in vitro. Ovaries were isolated from wisent females outside the reproductive season and eliminated from breeding for reasons other than infertility. Cumulus-oocytes complexes (COC) were isolated from all follicles above 2 mm in diameter. All COC were matured in TCM 199 supplemented with 10% fetal bovine serum, 0.02 IU mL–1 of porcine FSH, 17 β-oestradiol, 0.2 mM Na pyruvate, and antibiotics. The COC were cultured for 24 h at 38.5°C and 5% CO2 in humidified air. The matured COC (Bison bonasus) were fertilized in vitro with sperm from Jersey bulls (Bos taurus) in TALP supplemented with 6 mg mL–1 of fatty acid free BSA (BSA FAF), 0.2 mM Na pyruvate, 20 µM penicillamine,10 µM hypotaurine, 1 µM epinephrine, 2 µg mL–1 heparin, and antibiotics. Spermatozoa were used at a final concentration of 1 × 105 per oocyte and were co-cultured for 18 h at 38.5°C and 5% CO2 in humidified air. The hybrid zygotes were cultured in KSOM supplemented with 5 µg mL–1 of MEM Nonessential Amino Acid Solution (100×), 3 mg mL–1 of BSA FAF, and antibiotic for 192 h at 38.5°C and 5% CO2 in humidified air. The medium was partly replaced by fresh medium after 48 and 144 h of culture. Development was evaluated every day. From 25 COC isolated from wisent ovaries, only 18 COC were qualified for in vitro maturation (60%). Of these, 15 COC (83.3%) matured. The percentage of hybrid embryos that cleaved was 80% after 48 h of culture, and the percentage of embryos that developed up to the 8-cell stage was 33% after 96 h of culture. The morula/blastocyst rate was 26.6% after 192 h of culture, as represented by 1 early blastocyst, 2 compact morulae, and 1 morula. The use of the cattle IVP procedure allowed to receive hybrid embryos (Bison bonasus × Bos taurus), but they developed slower than cattle embryos under the same conditions, based on our previous studies. This research will be continued and may make a contribution to the protection of this threatened species.

The TSG-6 is a ~35-kDa protein belonging to hyaluronan binding superfamily proteins. The TSG-6 plays role in inflammation and in inflammation-like processes (i.e. ovulation). The tsg-6 expression is induced in cumulus–oocyte complex (COC) cells just before ovulation. It is involved in the migration of cumulus cells though the formation and stabilisation of the extracellular matrix. Disturbances in secretion of this protein lead to a reduction in the number of ovulated oocytes. Although studies of the prevalence and role of TSG-6 in COC were conducted in several animal models, little is known about TSG-6 in cattle. The aim of this study was to assess tsg-6 mRNA expression and protein localization in bovine cumulus cells from COC at different maturation status. Ovaries were collected from the slaughterhouse. Cumulus cells were isolated from COCs in different stages of maturity. In variant I, COC were isolated from small follicles of Φ 2 to 6 mm. In variant II, COC were also isolated from small follicles and matured in vitro in TCM199 HEPES with 10% FBS and 0.02 IU NIH-pFSH mL–1, 1 µg mL–1 β-oestradiol, 0.2 µM sodium pyruvate, 50 µg mL–1 gentamicin in 5% CO2, 38.5°C for 24 h. In variant III, COCs were isolated from large follicles of Φ >15 mm. Analysis of tsg-6 expression in cumulus cells was performed using real-time PCR. Expression of Tsg-6 was normalized to that of s18. Statistical analysis of tsg-6 mRNA level in all variants was carried out by 1-way ANOVA, and comparisons of mean values were made with the Tukey honestly significant difference test (Statgraphic 5.1 Centurion); P < 0.05 was considered to reflect the presence of statistical significance. For all variants, paraffin-embedded slices of COC were carried out to localise TSG-6 by indirect method of immunofluorescence. Immunostaining of the TSG-6 protein was performed using primary polyclonal antibody raised against bovine TSG-6. Antigen–antibody complexes were visualised after incubation with secondary IgG conjugated with fluorescein isothiocynanate. Immunolocalization of TSG-6 was performed using fluorescent microscopy. The relative expression of tsg-6 mRNA in variant II was more than 2 times higher (1.887 ± 0.797 a.u.) compared with variant III (0.760 ± 0.130 a.u.). The difference was statistically significant (P < 0.05). No tsg-6 expression in variant I was detected. The presence of TSG-6 protein in the extracellular matrix of cumulus cells from variant II as well as from variant III was detected. The present data suggest that tsg-6 gene expression and its protein presence are stimulated during COC maturation in cattle as previously demonstrated for other species. Supported by Warsaw University of Life Sciences, Faculty of Veterinary Medicine: 505-10-02330050.