This work was carried out to evaluate the quality and the developmental competence of the in vitro buffalo embryos produced under different culture conditions. Oocytes were matured in TCM 199 for 24 h at 38.5°C and 5% CO 2 . After maturation, oocytes were in vitro fertilized by frozen thawed sperms capacitated in vitro by caffeine at final concentration 2 million sperm /ml. The sperm-oocytes were incubated in TALP medium for 24 hrs. Fertilized oocytes were cultured in Cell free media (CR1aa) or with cumulus or oviductal cell monolayers (Experiment 1), CR1aa with addition of (10, 20, 50 and 100 µg/mL) lactoferrin (Experiment 2) inside CO 2 incubator with 5% CO 2 at 38°C. The cleavage, morula, blastocyst rates were recorded and embryo cell number and mitochondrial function were evaluated. Addition of bovine oviductal cell monolayer (BOCM) to culture media significantly increased the morula and the blastocyst compared with cell free media and media with cumulus cell monolayer and also significantly increased the cell number and the mitochondrial function of in vitro produced buffalo embryos. Addition of lactoferrin by concentration 50µg/ml to culture media resulted in a significant increase in rates of cleavage; morula and blastocyst compared with control or other concentrations and also resulted in an increase in the cell number and the mitochondrial function of in vitro produced buffalo embryos. It could be concluded that, addition of oviductal cells (monolayer) or lactoferrin to culture medium significantly improved the quality of in vitro produced buffalo embryos.

Infertility problem is a common syndrome among bitches and its causes are numerous, but the apparent most frequent cause is the mating at incorrect time. The aim of this study was to avoid the possible causes of infertility in bitches by predicting the optimal time of breeding and try to treat disorders in estrus cycles. A total number of 18 bitches of German shepherd breed were used in this study for monitoring phases of estrus cycle and predicting the optimal time of breeding. Those bitches examined by vaginal cytology, progesterone assay, measurement of vaginal PH, vaginal endoscopy and ultrasonographic examination of ovaries from proestrus to diestrus. By vaginal cytology, cornified cells first appeared on Day 4 from the beginning of proestrus, then increased till reached to 85% on Day 12 which is considered as the ovulation day. Measuring of progesterone concentration at the beginning of proestrus was very low (less than 1 ng/ml) then increased in 13 bitches only out of 18 bitches which used in this study till reach to 6.5 ± 1.37 ng/ml within ranges (4.8 to 8.3 ng/ml) on Day 12 from the beginning of proestrus. The other five bitches showed disorders in normal sequence of estrus cycle which was discovered by progesterone assay, vaginoscopy and ultrasonography of ovaries. Abnormalities in estrus cycle were recorded; two cases of anovulation and three bitches of persistent proestrus. As a trial for correcting the deviated estrus cycle, we injected hCG hormone in two anovulatory bitches for occurrence of ovulation, and also used methergine drug in cases of persistent proestrus as a trial for stoppage of continuous bloody discharge. The optimal time of breeding in 13 ovulatory bitches was within 2 to 3 days after ovulation day. Pregnancy rate after breeding by 20 days was recorded by ultrasonographic examination was 92.3 % (12/13 bitches). When applying these applications, we concluded that progesterone hormone assay was the accurate method for predicting the ovulation time and achieving higher pregnancy rate , and also can judge on estrus cycle if normal or abnormal.

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In the present study, seven mongrel bitches were used, their age ranged from 15 months to 3 years. Vaginal cytology was carried out during pregnancy period of these bitches by taking vaginal smears weekly to investigate vaginal cytological changes. Serial ultrasonography was performed daily on these bitches from the 15 th day after the last breeding until birth day to determine the time of appearance of different gestational structures. The first observation of the extra-fetal and fetal structures was as follows: The yolk sac membrane was first detected as an echogenic U-shaped fetal membrane in a range of 24-25 days. A third echogenic fetal membrane, allantoic membrane, was first detected on Day 27. On Day 23, in the uterine wall surrounding the gestational sac, an apparently hyperechoic inner layer was differentiated to the zonary placenta which appeared as two thick bands one on either side of the fetus on Day 29. The embryo appearance was on Day 20.5. The early time of the position of the embryo either opposed to the uterine wall or dependent on chorionic cavity was nearly on Day 22 and on Day 24, respectively. The heartbeat, which is one of fetal vital signs, was detected as a bright echogenic flickering motion on Day 21. On Day 26 in mongrel bitches, the features of the embryo changed from an oblong to bipolar shape and form two loops of number 8, size and density the same. An anechoic area in the head of the embryo was detected on Day 29. The limb buds were firstly detected on Day 29. Fetal movement was firstly detected on Day 33. Meanwhile, the skeleton of the fetus was first detected as hyperechoic structure on Day 33.