Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock.

Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus-oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.

Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes.

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells.

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

Physiology and Endocrinology Symposium: influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature.

High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation.

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.

Effects of retinoids on the in vitro development of Capra hircus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre-implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus-oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus-oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39 °C in an atmosphere of 5% (v/v) CO(2) in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39 °C in a humidified atmosphere of 5% (v/v) CO(2), 5% (v/v) O(2) and 90% (v/v) N(2). In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO(2) in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 µg/ml RT and 0.5 µm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre-implantation development of goat embryos and can be used to enhance in vitro embryo production.

Serum starvation and full confluency for cell cycle synchronization of domestic cat (felis catus) foetal fibroblasts.

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < or = 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < or = 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.

Oestrogen and progesterone receptor gene expression in canine oocytes and cumulus cells throughout the oestrous cycle.

The aim of this research was to analyze oestrogen receptor-alpha (ERalpha), ERbeta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells' total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ERbeta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ERbeta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ERalpha and ERbeta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ERalpha was expressed throughout the oestrous cycle.

Effect of maternal heat-stress on follicular growth and oocyte competence in Bos indicus cattle.

The objective was to determine whether exposure of Gir (Bos indicus) cows to heat-stress (HS) causes immediate and delayed deleterious effect on follicular dynamics, hormonal profile and oocyte competence. The cows were kept in tie-stalls for an adaptive thermoneutral period of 28 days (Phase I, Days -28 to -1). In Phase II (Days 0-28) cows were randomly allocated into control (CG, n=5) and HS (HS, n=5) treatments. The HS cows were placed in an environmental chamber at 38 degrees C and 80% relative humidity (RH) during the day and 30 degrees C, 80% RH during the night for 28 days. The CG group was maintained in shaded tie-stalls (ambient temperature) for 28 days. During Phase III (Days 28-147) animals were placed in tie-stalls (Days 28-42) followed by pasture (Days 42-147) under thermoneutrality. In each phase, weekly ovum pick up (OPU) sessions were to evaluate follicular development, morphology of cumulus-oocyte complexes (COCs), and developmental competence after in vitro maturation, fertilization, and culture. Serum concentrations of progesterone (P(4)) and cortisol were evaluated by radioimmunoassay. Exposure of Gir cows to HS had no immediate effect on reproductive function, but exerted a delayed deleterious effect on ovarian follicular growth, hormone concentrations, and oocyte competence. Heat-stress increased the diameter of the first and second largest follicles from Days 28 to 49. Indeed, HS increased the number of >9 mm follicles (characterized as follicular codominance) during this phase. Cows exposed to HS had longer periods of non-cyclic activity (P(4)<1 ng/mL), as well as shorter estrous cycles. However, HS did not affect cortisol concentration as compared to CG. Although HS had no significant effect on cleavage rate, it reduced blastocyst development during Phase III. In conclusion, long-term exposure of B. indicus cattle to HS had a delayed deleterious effect on ovarian follicular dynamics and oocyte competence.

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions.

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.

Manipulation of antioxidant status fails to improve fertility of lactating cows or survival of heat-shocked embryos.

Experiments were conducted to test whether enhancement of antioxidant status could improve fertility and milk yield in dairy cows and resistance of cultured embryos to heat shock. Three experiments in three herds were performed to evaluate the effect of multiple intramuscular injections of 500 mg of vitamin E and 50 mg of selenium at 8 to 21 d before expected calving and at 30 and 80 d postpartum on reproduction of lactating Holstein cows. Vitamin E and selenium injections did not improve reproductive function or milk yield in any of the studies. The predicted 305-d milk yield (averages of least-squares means across treatments) were: 9478, 7073, and 10,204 kg projected 305-d milk for experiments 1, 2, and 3, respectively. Percentages of cows pregnant at first service were 30, 16, and 24% in experiments 1, 2, and 3, respectively. Three studies were performed to test whether vitamin E improved development of cultured bovine embryos exposed to heat shock. Heat shock of 41 degrees C at the two-cell stage reduced development to the blastocyst stage, but culture with 100 microM vitamin E did not reduce effects of heat shock on impaired development. For example, 9 h at 41 degrees C reduced blastocyst development from 51.2 +/- 3.3% to 3.4 +/- 3.3% in the absence of vitamin E and from 54.0 +/- 3.3% to 5.2 +/- 3.3% in the presence of vitamin E. Development of morulae to the blastocyst stage was not compromised by culture at 41 degrees C for 9 h. Additionally, there was no overall effect of vitamin E on morula development. In conclusion, multiple injections of vitamin E and selenium at the administered levels did not improve postpartum fertility nor milk yield of lactating Holstein cows in three different herds, and there was no direct thermoprotective effect of vitamin E for cultured, heat-shocked embryos.

Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed, lactating cows.

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P < 0.05) and tended to increase pregnancy rate at d 81 (P < 0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P < 0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P < 0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P < 0.08) and the subset of synchronized recipients (P < 0.10). There were no effects of GnRH (P > 0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P > 0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.

Genetic divergence in cellular resistance to heat shock in cattle: differences between breeds developed in temperate versus hot climates in responses of preimplantation embryos, reproductive tract tissues and lymphocytes to increased culture temperatures.

The detrimental effects of heat stress on fertility in cattle are less pronounced in heat-tolerant breeds. Although these genetic differences reflect differences in thermoregulation, cells from heat-tolerant breeds are less adversely compromised by increased temperature (that is, heat shock) than cells from heat-sensitive breeds. Experiments were performed to test the hypothesis that cells and tissues from two thermotolerant breeds (Brahman and Senepol) are better able to survive and function after exposure to increased temperature than cells and tissues from two thermosensitive breeds (Holstein and Angus). Exposure of embryos at>eight-cell stage at day 5 after insemination to heat shock of 41.0 degrees C for 6 h decreased development to the blastocyst stage and the number of cells per embryo. However, the deleterious effect of heat shock on blastocyst formation and the number of cells per embryo was less pronounced for Brahman than for Holstein and Angus breeds. Embryos from Senepol cows had very low development and it was not possible to determine heat shock effects in this breed. In contrast to the sensitivity of embryos to heat shock, there was no effect of a 41.0 degrees C heat shock on [(3)H]leucine incorporation into proteins secreted by oviductal or endometrial explants. Lymphocytes from Brahman and Senepol cows were more resistant to heat-induced apoptosis than lymphocytes from other breeds. Heat shock reduced lymphocyte glutathione content but the magnitude of the decrease was not affected by breed. In conclusion, embryos from Brahman cows are more resistant to heat shock than embryos from Holstein or Angus cows. Genetic differences are also present in thermotolerance for apoptosis response in lymphocytes, with Brahman and Senepol cattle being more resistant to heat shock than Angus and Holstein breeds. It is likely that the evolutionary forces that led to the Brahman and Senepol breeds being adapted to hot climates resulted in the selection of genes controlling resistance to cellular heat shock.

Apoptosis is an adaptive response in bovine preimplantation embryos that facilitates survival after heat shock.

Heat shock compromises development of preimplantation bovine embryos and the percentage of blastomeres labeled as TUNEL-positive. It was hypothesized that TUNEL labeling represents apoptosis and that apoptosis after heat shock is beneficial for continued embryonic development. To test these hypotheses, experiments were performed with z-DEVD-fmk, an inhibitor of group II caspases, on heat shock responses of embryos > or =16-cell stage at day 4 after insemination. Heat shock of 41 degrees C for 9 h increased group II caspase activity and the proportion of TUNEL positive cells; z-DEVD-fmk blocked these effects. The reduction in development of embryos exposed to heat shock for 6-9 h was magnified in the presence of z-DEVD-fmk. Results indicate that group II caspases mediate heat-induced apoptosis in bovine embryos and that inhibition of these caspases has a detrimental effect on embryonic resistance to heat shock. Apoptosis can be viewed as an adaptative mechanism to allow embryonic survival and development following stress.

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos.

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.

Effect of season and exposure to heat stress on oocyte competence in Holstein cows.

Two experiments were conducted to evaluate seasonal variation in oocyte competence in Holstein cows and to test whether oocyte quality in summer is affected by the magnitude of heat stress. In the first experiment, ovaries of Holstein cows were collected from a slaughterhouse and used to harvest oocytes over 1 yr (n = 18 replicates). After in vitro maturation, fertilization, and culture, proportions of oocytes and cleaved embryos that developed to blastocysts by d 8 were lower in the warm season compared with the cool season. In the second experiment, nonlactating Holstein cows were housed in one of the following three environments for 42 d before slaughter: heat stressed (housed with shade cloth in summer; n = 14); cooled (housed in a free-stall barn with foggers and fans in summer; n = 14); and winter (housed similar to the heat-stressed group; n = 12). Cows were slaughtered at d 18 to 19 of the estrous cycle. Oocytes from the two largest follicles per cow were aspirated and cultured individually. Ovaries were then dissected to collect additional oocytes that were processed in a group for in vitro maturation, fertilization, and culture. Cleavage rates were similar among treatments, but none of the individually cultured oocytes developed to blastocysts. For other oocytes cultured in groups, proportions of oocytes and cleaved embryos that developed to blastocysts by d 8 were lower in summer than winter with no difference between the heat-stressed and the cooled treatment groups. Summer depression in oocyte quality in Holstein cows was evident, but cooling cows for 42 d did not alleviate that seasonal effect.

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon.

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.