In in vitro experiments, the percentage of bovine oocytes that develops to the blastocyst stage is much lower compared with the in vivo counterparts. The quality of the oocyte is the main factor affecting blastocyst yield. Moreover, in vitro-produced bovine embryos are more sensitive to cryo-injuries than those produced in vivo. Exposure of oocytes to sub-lethal concentrations of stressors may enhance their quality through upregulation of intracellular shock proteins. We aimed to evaluate whether addition of ethanol at low concentrations (0.27 or 0.53%) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus-oocyte complexes (n = 934) were matured in serum-free TCM199 plus 20 ng mL-1 of epidermal growth factor (control), supplemented with ethanol 0.27% (treatment 1) or 0.54% (treatment 2), in 3 replicates. After fertilization, the presumptive zygotes were cultured for 6 days in modified SOF medium supplemented with 5% fetal calf serum (FCS); the number of blastocysts was recorded and classified. Then, expanded blastocysts were cryopreserved by open pulled straw vitrification using the 2-step approach described by Vajta G et al. (1998 Mol. Reprod. Dev. 51, 53-58). After 24 h, vitrified embryos were warmed and cultured in groups of <25 per 50 μL droplet of modified SOF medium with 5% FCS under mineral oil for 48 h and examined for re-expansion and hatching. Differences between the groups in blastocyst yield were analyzed by ANOVA; differences in survival rates between the groups were analyzed using logistic regression analysis. For all statistical models, group was included as fixed effect, and also the effect of replicate was included. Differences were considered to be statistically significant when P < 0.05. Addition of ethanol to in vitro maturation media had no significant effects on blastocyst yield on 7 dpi. Also, addition of ethanol at 0.27% did not affect blastocyst cryotolerance. However, addition of ethanol 0.54% to in vitro maturation media significantly increased the survival of bovine blastocysts after vitrification (P < 0.01; Table 1). The results of the present study indicate that maturation of oocytes under ethanol stress at low concentrations has carry-over effects on embryo quality, leading to improved cryotolerance. Table 1.Survival percentage (mean ± SE) of vitrified expanded bovine blastocysts matured in the presence of sub-stress concentrations of ethanol The authors thank I. Lemahieu and P. Vandamme for their excellent technical support. This research was supported by the special research fund, Ghent University (Grant, BOF/DOS No. 01W05706).

Metabolic profile changes of the follicular fluid of the growing follicles can be used as an indirect indicator of the oocyte and granulosa cells quality. The aim of this study was to investigate the biochemical compositions of follicular fluid collected from follicles at different stages of growth and their relationship with that of blood serum in Dromedary camel. Ovaries were colleceted from local slaughterhouse (Oniza, KSA). Soon after slaughtering, blood samples were collected from Dromedary camel (n = 20) and follicular fluid was aspirated from three different groups of non-atretic follicles (4&ndash;6)mm, 6&ndash;8mm and 10&ndash;20mm diameter). Follicular samples were pooled by maintaining the follicular sizes. Concentrations of glucose, cholesterol, triglycerides, urea, total protein, lactate dehydrosenase (LDH), cortisol, triiodothyronine (T3), insulin-like growth factor-1 (IGF-1) and non-esterified fatty acids (NEFA) were assayed in each serum and follicular fluid sample. The concentrations of glucose, cholesterol, triglycerides, total protein, LDH, T3, IGF-1 and NEFA decreased in the follicular fluid irrespective of follicular sizes and increasing in trend for urea in comparison with blood serum. There was a significant concentration gradient for IGF-1 in small follicular group compared to large or medium groups. Our data from the present study suggest that the oocyte and the granulosa cells of Dromedary she camel develop in a biochemical environment that does not have remarkable changes from small to large follicles except for IGF-1. In conclusion, the above-mentioned metabolic changes in the growing follicle is related to blood metabolic changes and, therefore, may be used in determining follicular dominance, oocyte and granulosa cells quality in Dromedary camel.

High concentrations of non-esterified fatty acids (NEFA) during negative energy balance (NEB) in high yielding dairy cows have been proven to be partially responsible for reduced fertility. This hypothesis has been tested by the addition of NEFAs to in vitro maturation medium at concentrations present in follicular fluid during NEB. We aimed to evaluate whether high concentrations of palmitic acid (C16:0) (PA), stearic acid (C18:0) (SA), or oleic acid (C18:1) (OA) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus–oocyte complexes (n = 4600) were matured in serum-free TCM199 plus epidermal growth factor (EGF, 20 ng mL–1; negative control), supplemented with ethanol alone (positive control) or with 0.133 mmol L–1 PA, 0.067 mmol L–1 SA, or 0.200 mmol L–1 OA (NEFAs dissolved in ethanol). The three NEFAs were tested separately in 4 replicates for PA and 5 replicates for OA or SA. Each fatty acid tested per replicate including a negative and a positive control group. After the embryos were cultured for 7 days in SOF medium, the number of blastocysts was recorded and classified as expanded, hatching, or hatched. Then, blastocysts were cryopreserved by open pulled straw vitrification using the two-step approach described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Vitrified warmed embryos were cultured in groups of <25 per 50-μL droplet of modified SOF medium with 5% fetal calf serum (FCS) under mineral oil for 48 h and examined for re-expansion and hatching. The percentages of survival in the different treatment groups were analyzed using logistic regression analyses, including the effect of replicates. Survival or not was included as the dependent variable and group was the independent variable. For every fatty acid a separate model was used. For all analyses, differences were considered to be statistically significant at the P < 0.05 level. Addition of OA to in vitro maturation media had no significant effects on cryotolerance of embryos. However, addition of PA or SA to in vitro maturation media (Table 1) significantly (P < 0.05) decreased the survival of bovine blastocysts from 79% in the positive control to 57% in PA and from 61% to 53% in SA. The results of the present study indicate that maturation of oocytes in the presence of NEB-associated concentrations of PA and SA can have carry-over effects on embryo quality, leading to reduced cryotolerance. We suggest that elevated NEFA concentrations in the follicular fluid may be one of the factors through which NEB exerts its negative effects on fertility in high yielding dairy cows. Table 1. Survival percentage (mean ± SD) of vitrified expanded bovine blastocysts matured in palmitic acid (C16:0) or stearic acid (C18:0) The authors thank J. Mestach and G. Spaepen for their excellent technical support. This research was supported by the Ministry of the Flemish Community, Belgium, in cooperation with the Ministry of Higher Education, Egypt.

The hyperthermia associated with summer heat stress (HS) is a major cause of reduced fertility in high yielding dairy cows in the tropics and subtropics. The hyperthermia directly affects reproductive function. Furthermore, HS induces indirect effects (e.g. through a dropped dry matter intake and thus an altered energy balance), which may also have an impact on cow fertility. The extent of exposure to HS and HS-associated metabolic changes, such as hypoglycemia and high levels of non-esterified fatty acids (NEFA), may affect cattle fertility by impairing the developmental capacity of the oocyte. It is known that changes in glucose and NEFA concentrations in the blood may be reflected in the follicular fluid of the dominant follicle (Leroy et al. 2004). Therefore, the aim of the current study was to estimate to what extent oocytes may be exposed to HS-associated metabolic changes in dairy cattle during summer by assessing the diurnal changes in plasma. Respiration rate (RR) and rectal temperature (RT) along with plasma concentrations of glucose and NEFA were examined in 10 lactating Holstein cows at 4-h intervals from 7:00 AM to 3:00 AM for three successive days in August 2007. Temperature humidity indices (THI) were calculated as well. Linear mixed effect models with cow as random factor including the effect of daytime were used to investigate possible differences among studied parameters at different daytimes. At 3:00 PM, THI and RT were the highest (83.7 ± 0.6 and 40.1 ± 0.1, respectively) and the lowest value for THI recorded at 3:00 AM (74.2 ± 1.4) and for RT recorded at 7:00 AM (39.04 ± 0.08). RR increased gradually from 66.1 ± 3.1 resp min–1 at 3:00 AM to 93.0 ± 4 resp min–1 at 11:00 AM. Glucose concentrations decreased significantly (P < 0.001) at 11:00 AM and 3:00 PM. Decreased glucose concentrations coincided with increased NEFA concentrations at 11:00 AM and 3:00 PM (P < 0.001). The NEFA concentrations measured at 3:00 PM were significantly higher than at all other time points (Table 1). In conclusion, high producing dairy cows may be exposed to diurnal fluctuation of HS-associated metabolic changes such as significantly decreased glucose and increased NEFA concentrations in plasma for about 12 h per day, which may be reflected in the follicular fluid of the dominant follicle and as such directly affect oocyte and granulosa cell quality. Table 1.Mean ± SEM of glucose and non-esterified fatty acids (NEFA) concentrations (mmol L–1) in blood plasma of 10 lactating Holstein cows at 6 different daytimes during August 2007 in Egypt