Heat stress is one of the main reasons for reproductive performance decrease in cattle, resulting in severe economic losses. The aim of this study was to evaluate the effect of heat stress during maturation, fertilization and development of in vitro produced bovine embryos. Cumulus oocyte complexes (COCs) were obtained by follicular puncture from slaughterhouse ovaries and after identification, were divided into four groups: control (CG), exposed 1 (EG1), exposed 2 (EG2), and exposed 3 (EG3). The oocytes of the group CG and CG3 were cultured at 38°C and the oocytes of group EG1 and EG2 were cultured at 40°C during the maturation period (24 hours at 5% CO2 in air). After the maturation period, oocytes of group CG, EG1, EG2, and EG3 were fecundated with frozen thawed semen. The oocytes of CG, EG2 and EG3 groups were cultured at 38°C, and the group EG1 was cultured at 40°C (18 hours at 5% CO2 in air). After that, the CG and EG2 groups were cultured in SOF at 38°C and the groups EG1 and EG3 at 40°C during embryonic development. The embryos were evaluated for cleavage, morula and blastocyst rates by optical microscopy. In control (CG) and EG3 groups, the oocytes showed uniform expansion of cumulus cells, classified as moderate to high, with brown color and uniform appearance of the ooplasm. In the oocytes exposed to 40°C (EG1 and EG2) we observed a decrease in the expansion of cumulus cells, and the same showed rounded appearance and retraction of the ooplasm with dark coloration. The control group (CG) had 68.23% ± 2% of cleavage, 50.16% ± 2% morulas, and 43.28% ± 1% blastocysts. Whereas the EG2 had 31.46% ± 2% cleavage, 35.64% ± 2% morula, and no blastocysts development. The EG3 had 3.7% ± 2% cleavage, and no embryo production. These data suggest that in all stages of exposure to heat stress, the embryos and the gametes are susceptible, leading to a decrease in embryonic development.
There is a change in behavior as well as neuroendocrine and physiological responses to keep homeostasis for temperature adaptation in which the animal is being submitted. Regarding reproduction, during heat stress, the gonadotropins and gonadal hormones are changed, impairing the reproductive cycle. Given these changes, it is common to see the occurrence of reduced fertility, low rates of estrus identification, conception decrease, abortion and embryonic mortality [
On the other hand, the reproductive capacity is changed also by the direct action of raising the internal temperature of the animal reproductive tract cells and tissues [
According to Hansen and Aréchiga [
In general, the period of greatest vulnerability of the embryo in relation of heat stress for the establishment and maintenance of pregnancy is up to 4 - 8 cells. This affects the development to the blastocyst stage. However, when stress is focused at the morula stage no changes occur [7,8]. Pires et al. [
According to Edwards and Hansen [
The aim of this study was to evaluate the effect of heat stress during maturation, fertilization and development of in vitro produced bovine embryos.
For this study, oocytes were divided into four groups: control group (CG); exposed group 1 (EG1), kept at 40˚C during the maturation, fertilization, and embryonic development; exposed group 2 (EG2), kept at 40˚C during the oocyte maturation; exposed group 3 (EG3), maintained at 40˚C during the stage of embryonic development. All groups were evaluated for morphology, fertilization and embryo development rates.
Unless otherwise stated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tissue culture media 199 (TCM 199 Hepes and Bicarbonate-buffered), PBS, and fetal calf serum (FSC) were obtained from GibcoTM, Invitrogen Corporation (Grand Island, NY, USA).
Cattle ovaries were obtained from a local slaughterhouse (unknown breed), and transported to the laboratory in PBS with penicillin (100 units/mL)—streptomycin (100 μg/mL) at 38˚C. Cumulus oocyte complexes (COCs) were aspirated from 2 to 6 mm in diameter follicles, and selected only those had intact zona pellucid with uniform ooplasm surrounded by at least two layers of cumulus cells. All groups (CG; EG1; EG2; EG3) were matured (20 COCs/drop) in 100 μL of TCM-199, LH (6 μg/mL), FSH (8 μg/mL) (Sioux Biochemical, Sioux Center, IA, USA), and penicillin-streptomycin for 24 h at 38˚C (CG; EG3) and 40˚C (EG1; EG2), 5% CO2 in air [
Frozen-thawed spermatozoa (Bos indicus) were washed by a 45%/90% layered Percoll gradient centrifugation. Oocytes were co-incubated with 10 × 104 spermatozoa in the fertilization medium supplemented with 2 μg/mL of heparin and 20 μL of PHE solution (20 μM penicillamine, 10 μM hypotaurine, 1 μM adrenaline; Hasler et al., 1995). The control group (CG), EG2, and EG3 were cultured at 38˚C and and EG1 at 40˚C, 5% CO2 in air (v/v).
After 18 h, presumptive zygotes were washed, removing the cumulus cells by pipetting, and cultured with synthetic oviductal fluid (SOF; [
Each experiment was repeated five times and data from each experiment were pooled. Approximately 90 - 100 oocytes were evaluated in each replicate. Data were analyzed using least square means to determine the effect of each treatment on embryonic cleavage and development to the blastocysts stage (SAS Institute Inc., 1989- 1996). The significance level for all tests was P < 0.005.
After 18 h, the control group showed symmetrical cleavage and uniform ooplasm. The EG1 group, which was exposed at 40˚C during maturation, fertilization and embryo development, had retracted ooplasm with granular appearance. In the EG2, exposed at 40˚C during in vitro maturation, observed asymmetric cleavages, and retracted ooplasm with granular appearance. Whereas the EG3 group, exposed at 40˚C the embryo development, showed degenerated embryos with retracted ooplasm (
After the day 7, embryos from the control group (CG) were in morula and blastocysts stages. The EG1 and EG3 groups did not show morula and blastocysts, but the EG2 had some morulas and no blastocysts.
The
The interaction between animal and environment should be considered when seeking greater efficiency in the livestock exploration, due to the different responses of the animal to the peculiarities of the surrounding en-
vironment. Thus the correct identification of the factors that influence the productive life of the animal, like heat stress, result of climate change over the past year, may allow adjustments in production management, enabling them to sustainability and economic viability. Therefore, the need for a deeper and more detailed study of the damages caused by the increase of environmental temperature on animal reproduction, led us to this research in order to study the effect of heat stress during maturation, fertilization and development of in vitro produced bovine embryos.
In the present study, the control group (CG) showed 68.23% ± 2% of cleavage, 50.16% ± 2% morulas, and 43.28% ± 1% blastocysts. Whereas the oocytes exposed at 40˚C during maturation, fertilization, and embryo development (EG1) interfered with oocyte viability, inhibiting the process of fertilization and cleavage in 100%. However, in the EG2 (exposed at 40˚C during maturation), observed 31.46% ± 2% cleavage, 35.64% ± 2% morula, and no blastocysts development.
Edwards and Hansen [
Ju et al. [
These results support the hypothesis that oocytes exposed to heat stress and suffered cell damage may interfere with subsequent fertilization and embryo development, and that the longer the duration of exposure to high temperatures, the greater the damage. However, it was observed that the EG1 from this study, after 18 hours exposed at 40˚C, presented sperm with no mobility, cloggy and no tail. This shows that the increase in temperature adversely affects the survival of the spermatozoa and in the fertilization.
According to Hansen et al. [
Regarding EG3 group, exposed at 40˚C during embryo culture, was observed only 3.7% of cleavage, without the development of morulas and blastocysts. Similar results were described in an experiment conducted by Al-Katanani and Hansen [
Edwards and Hansen [
These findings may be due to the fact that the first cleavages correspond to the critical phase, in which genomic activation occurs through the maternal control to embryonic. During this period, there seems to be susceptibility to the heat stress, leading to failure in embryonic genome activation and blastocysts development [
According to Krininger III et al. [
Some studies demonstrated that the 2 cells bovine embryos, when subjected to heat stress at 40˚C for 3 hours, developed to the blastocysts stage normally [20,26]. The same authors observed that embryo development decreased when subjected to heat stress at 41˚C and 42˚C for a long period of time (12 hours). These effects were also reported in porcine embryos by Ju and Tseng [
Studies carried out by Bényei and Barros [
The authors thank the staff at the Fenix slaughterhouse, to FAPESP (2010/01077-9), Vitrocell/Embriolife®, and to CNPq.