Effects of Dietary Urea on timing of embryo cleavages and blood components in Mice

The objectives of the present study were to investigate the effects of dietary urea supplementation (1.0% and 3.0%) on oocytes quality, timing expected of embryo cleavages, offspring numbers and weights, blood components and rectal temperature in mice. Sixty of growing albino mice were classified into three groups; the control group was given basal control diet and the other two groups were fed on basal control diet supplemented with 1.0% and 3.0% urea. Body weights were recorded at the beginning and after 5 weeks. Thereafter, five female mice of each group were injected with 7.5 IU of pregnant mare serum gonadotropin (PMSG) for determination of oocyte quality after 48h of injection. The fifteen female mice of each group were injected with 7.5 IU of PMSG followed by 7.5 IU of human chorionic gonadotropin (hCG) after 48h and mated with males of proven fertility. Five mated females of each group were used for determination of embryo cleavages to four cell stage and the other five mated females were used for determination of embryo cleavages to eight cell stage upon 59-60 and 70 h of hCG injection, respectively. Rectal temperatures were recorded and blood samples were collected. The remaining five mated females of each group were left for parturition. The offspring number, litter size and male:female ratio were recorded. Hematocrit and hemoglobin concentrations were determined in blood whereas urea, total protein, albumin, glucose calcium and phosphorus concentrations were determined in plasma. The results indicated that offspring number and weight of litter size at birth were significantly (P<0.05) increased in the urea groups compared to control group. Percentage of good quality oocytes was high (70%) in control group compared to 3% urea group (60%). Dietary 3% urea was delayed cleavages to four-cell stage embryos at the expected time. Dietary urea was significantly (P<0.05) increased concentrations of hematocrit and hemoglobin in blood and urea, total protein, globulin, glucose, potassium and phosphorus in plasma. In conclusions, although 3% dietary urea decreased oocytes quality and timing expected of embryo cleavages to four cell stages, it increased significantly (P<0.05) offspring number and weight of litter size.


Introduction
embryos were recovered from superovulated donor mice fed diets supplemented with 1.0% and 3.0% urea.Most previous reports have provided evidence The collected oocytes and embryos were assessed for that high protein diets, which result in elevated levels quality and stage of cleavages respectively. of plasma urea nitrogen, are related to decreased In addition, offspring numbers and weights were fertility in ruminants (McEvoy et al., 1997;Dawuda et recorded of the mated females.Blood samples were al ., 2002).The timing and mechanism(s) underlying collected for analysis in addition to measuring rectal the deleterious effects of excessive urea nitrogen on temperature.fertility remain unclear.
The hypothesis that elevated plasma urea nitrogen Studies have examined the effects of urea concentrations would decrease embryo viability and nitrogen on oocyte maturation and embryo developresult in fewer pregnancies.ment in vitro (De Wit et al., 2001;Ocon and Hansen, 2003).The study of Rhoads et al., (2006) demons-

Materials and methods
trated that the viability of the embryo is altered before Animals and feeding: Sixty female albino mice (5 day 7 of pregnancy in dairy cattle.High levels of weeks of age) were used during the study for two plasma urea nitrogen changed the follicular, oviductal months and classified into three groups.The control and (or) uterine environment, which impacted the group was given basal control diet and the other two competency of the embryos for continued developgroups were fed on basal control diet supplemented ment beyond day 7.In the present study, oocytes and with 1.0% and 3.0% urea.All mice were allowed free were punctured by 30-ga needles, and cumulus -GV access to the experimental diets and water throughout oocyte complexes were released into Ringer's solutions supplemented with 10% serum.Germinal the experimental period.Food and water were vesicle oocytes were aspirated immediately by glass available ad libitum.Mice were killed by cervical pipette, the tip diameter of which was larger than the dislocation for collection of oocytes and embryos.
diameter of cumulus-enclosed oocytes and graded into Composition of the experimental diets is shown in Table 1.
good, fair and denuded oocytes.and before mating (after 5 weeks of starting).Weights Fifteen females of each groups were superovulated by of litter sizes were recorded upon deliveries.The body injection of 7.5 IU of PMSG followed by 7.5 IU of weight of animals was measured to the nearest ± 1.0 g human chorionic gonadotrophin (hCG; Chorulon, using a standard balance.Rectal temperature was Intervet) 48 h later and mated with males of proven measured using a digital clinical thermometer.
fertility.Ten females of each group were used for Blood Samples : Blood samples were collected of ten collection of cleaving embryos from the oviducts female mice in each group before collection of 59-60 and 70h h after hCG injection and the stages of cleaving embryos from the orbital sinus according to embryos were recorded (Grabrek et al., 2004;the method described by Hoff (2000).Hematocrit and Mohammed et al., 2010) whereas the remaining five hemoglobin concentrations were determined immefemales of each group were left for parturition.Weight diately in blood sample, then, the same remaining of litter size and number and male/female ratio were blood sample was centrifuged at 5000 rpm for 15 min recorded upon parturition.for obtaining plasma and stored at -20 °C until further Blood analysis : Blood samples were analyzed for analysis.
determination of hematocrit and hemoglobin concen-

Collections of germinal vesicle oocytes and
trations whereas plasma samples were analyzed for embryos: Germinal vesicle (GV) oocytes were collected from the five matured female mice in each determination of urea, total protein, albumin, glucose, group.The female mice were injected with 7.5 IU of potassium and phosphorus concentrations by using appropriate commercial test kits supplied by Spectrum pregnant serum gonadotrophin (PMSG; Folligon, Intervet).Ovaries were removed from the donor Diagnostics (Cairo, Egypt).The concentrations were females 44-48 h after PMSG injection.Antral follicles measured using standard protocols.

Discussion
Statistical analysis: Data are presented as means ± SD.Differences between mean values were determined Dietary urea levels (1 & 3%) were adversely by ANOVA followed by comparisons using the affected on oocyte quality and delayed timing of Duncan's multiple range test.Differences with P < 0.05 embryo cleavages at four cell stages.Developmental were considered significant.competence of embryos was restored at eight cell stage.Thereafter, weights of litter size at birth and

Results
offspring numbers were significantly increased.Dietary Effects of dietary urea level on body weight gain, urea supplementation was changed blood components weights of litter sizes and numbers and male/female where hematocrit, hemoglobin, urea, total protein, ratio are presented in table (2).Mating body weights globulin, glucose, potassium and phosphorus concen-(g) were significantly (P <0.05) decreased in urea trations were increased.groups compared to control whereas weights of litter Mating body weights were significantly decreased size at birth and offspring numbers were significantly in urea groups.Although the formulation of experim-(P <0.05) increased in urea groups.Male to female ental diet supplied sufficient amounts of nitrogen, ratio was not differed among groups.Percentage of energy, minerals and vitamins (table 1) in urea groups good oocytes was decreased (60%) in urea (3%) group and control group, this might be due to poor compared to urea (1%) and control (70%) groups palatability of urea which reduced dry matter intake.(table 3).Furthermore, effects of dietary urea level on The adverse effects of dietary urea supplemtiming of embryo cleavages collected 59-60 h and 70h entation (1 & 3%) on oocyte quality in mice were after hCG injection were presented in table (4).Urea consistent with our results in sheep (Mohammed et al.,levels (1 & 3%) were delayed timing of embryo unpublished).Moreover, dietary urea levels (1 & 3%) cleavages at the expected time.The percentage of two were associated with delayed embryo cleavages at the cell embryos found at the expected four-cell stage proper time.In other studies, dietary urea levels were embryos was higher (23.4 & 26.6%) in urea groups associated with decreased embryo quality (McEvoy et compared to control (12.5%) indicating delayed al., 1997).Thereafter, the embryos were restored cleavages of embryos.The percentage of four cell developmental competence.Our results (Mohammed embryos found at the expected eight-cell stage et al., 2005) concerning the effect of adding bovine embryos was comparable among groups indicating follicular fluid to the maturation medium indicated restoring development.Rectal temperatures were not that timing of first cleavage was delayed.In addition, differed among groups (table 5).Dietary urea levels (1 developmental competence to the blastocyst stage was and 3%) were significantly (P<0.05)increased significantly increased compared to adding of serum.hematocrit, hemoglobin, urea total protein, globulin, Therefore, the changes of follicular fluid compoglucose, potassium and phosphorus.(table 5).
sitions might be responsible for the delayed timing of Body weight and rectal temperature : The body Recording stage of cleaving embryos, weight of weights were recorded at the starting of experiment litter size and number and male/female ratio :

Table - 2. Effects of dietary urea level on body weight (BW), litter size and number and male/female ratio
a, b: Values with the different superscripts in the same row differ significantly (P<0.05).

Table - 5. Effects of dietary urea level on rectal temperature and blood components
Values with the different superscripts in the same row differ significantly (P<0.05).