Basic Informations

C.V

Visiting Scientist, Shanghai Institute of Biochemistry and Cell Biology, China, 2014

(Award of Chinese Academy of Sciences)

Visiting Fellow, Central Institute for Research on Buffaloes (CIRB), India, 2012

(Award of CV Raman Fellowships for African Researchers)

Master Title

Cytogenetic Studies on Cattle and Buffaloes in relation to cetain infertility Problems

Master Abstract

SUMMARY Ovaries were collected from apparently healthy slaughtered female buffaloes and after gross genital findings of the abattoir ovaries, they were classified according to the age and fertility status of slaughtered buffaloes and morphological aspects of these ovaries into four groups as follow: non-aged fertile ovaries having functional structures (group 1), non-aged infertile having pathological or no structures (group 2), aged fertile having functional structures (group 3) and aged infertile having pathological or no structures (group 4). At the laboratory, for each group, the number of the follicles (2-6 mm) on the ovaries was counted and then the follicular contents were collected from ovarian follicles by aspiration method and then the recovered oocytes were classified and counted for each group. The good quality recovered oocytes were matured in two models. Model 1: TCM-199, FCS, PMSG and hCG Model 11: TCM-199 and FCS only and then incubated for 20 hours at 39 ºc in atmosphere containing 5 % CO2 and 95 % relative humidity . The incidence of matured oocytes and their quality were recorded for each case. Matured oocytes were cocultured with spermatozoa capacitated in two models , Model 1 contain TALP and Model 2 contain BO medium containing caffeine and heparin for 10 hours in TALP or 6 hours in BO hours .then they were cultured in TCM -199 for 48 hours. The incidence of cleaved oocytes were recorded for each case The in vitro produced embryos after 48 hrs of insemination were cultured in two models, Model1 (TCM-199 supplemented with 10% FCS and cumulus cells) and Model 11 (TCM-199 supplemented with 10% FCS). The incidence of development into blastocyst was recorded for each case. The cryopreserved immature and mature buffalo oocytes and buffalo embryos were thawed and then survival rate for each case , then complete development of immature and mature oocytes on Model 2 of IVM / IVF / IVC technique . Some of matured oocytes and buffalo embryos were subject to cytogenitical analysis. The results of the present study indicated that: 1- Oocytes recovery yields of the four ovarian groups 1.1-Recovery of immature buffalo oocytes The total number and the average number of the oocytes recovery per ovary collected from four studied groups of buffalo ovaries are listed in tables (1, 2 & 3) and figure (5). When the 4 groups of buffalo ovaries were compared for the average number of oocytes recovery per ovary (Tables 1, 2 & 3), the non-aged fertile ovarian group 1 yield a higher number of oocytes per ovary (p<0.01) than that of aged fertile ovarian group 3, non-aged infertile ovarian group 2 and aged infertile group 4 (2.9 + 0. 33 vs 2.5 + 0.34, 1.37 + 0.13 and 1.28 + 0.16, respectively). Moreover, when the last three groups were compared, the aged fertile group 3 yield a higher number of oocytes per ovary (p<0.01) than that of non-aged infertile group 2 and aged infertile group 4 (2.5 + 0.34 vs 1.37 + 0.13 and 1.28 + 0.16, respectively). Moreover, the differences between the values of non-aged infertile ovarian group 2 and aged infertile ovarian group 4 were statistically significant, p<0.05, (1.37+0.13 vs 1.28+0.16, respectively). 1.2 - Quality of recovered buffalo oocytes The recovered oocytes in the present study were classified into three quality (Fig. 6) which included CCOCs, SCOCs and Nude. With regard to the quality of recovered oocytes, tables (1, 2 & 3) and figure (5) showed the total and average number as well as the percentage of the three classes of recovered buffalo oocytes collected from four studied groups of buffalo ovaries. The average number of CCOCs per ovary was significantly higher (p<0.01) in both non-aged fertile and aged fertile ovarian groups (0.79 + 0.27 and 0.54 + 0.03, respectively) than non-aged infertile and aged infertile ovarian groups (0.23 + 0.06 and 0.14 + 0.04, respectively). The present study revealed also that average number of SCOCs per ovary was significantly higher (p<0.01) in both non-aged fertile and aged fertile groups (1.2+0.2 and 1.19+0.42, respectively) than non-aged infertile and aged infertile groups (0.439+0.11 and 0.47+0.18, respectively) . Concerning values of the average number of Nude oocytes per ovary was significantly higher (p<0.01) in both non-aged infertile and aged infertile groups (0.65+0.07 and 0.65+0.37, respectively) than those of the non-aged fertile and aged fertile groups (0.37 + 0.19 and 0.49 + 0.37, respectively). 1.3 - Cultured buffalo oocytes The present results (Tables 1 , 2 & 3 and Fig. 3), revealed that both the average number of the cultured oocytes (CCOCs and SCOCs) per ovary for the non-aged fertile ovarian group 1 was significantly higher than those of groups 3 , 2 and 4 ( 2.19 + 0.38 vs 1.78 + 0.14 , 0.66 + 0.07 and 0.64 + 0.39 , respectively ) Moreover, when the latter three groups were compared , the aged fertile ovarian group 3 yield a higher number of oocytes per ovary than each of non-aged infertile ovarian group 2 (p<0.01) and aged infertile ovarian group 4, p<0.05 (1.78+0.14 vs 0.66+0.07 and 0.64+0.39, respectively). The results of aged infertile group 4 revealed a non-significant lower yield of oocytes suitable for maturation than that of Non-aged infertile group 3 (0.64 + 0.07 vs 0.66 + 0.07, respectively). 2- Oocytes developmental competence of the four ovarian groups 2.1- Matured buffalo oocytes (IVM - Model 1) The statistical analysis among the four ovarian groups revealed that the non-aged fertile ovaries group 1 gave a higher number of matured oocytes per ovary (p<0.01) than that of aged fertile ovarian group 3 , non-aged infertile ovarian group 2 and aged infertile ovarian group 4 (1.79+0.37 vs 1.25+0.11, 0.35+0.11 and 0.36+0.16, respectively). Moreover ,when the last three groups were compared , the aged fertile ovarian group yield a higher number of matured oocytes per ovary (p<0.01) than that of non-aged infertile ovarian group 3 (p<0.01) and aged infertile ovarian group 4, p<0.05 (1.25+0.11 vs 0.35+0.11 and 0.31+0.16, respectively). The aged infertile ovarian group 4 resulted in a non-significant lower in maturation rate than that of non-aged infertile ovarian group 2 (0.35 + 0.11 vs 0.31+0.16, respectively). Quality of matured buffalo oocytes The matured oocytes in the present study were classified into three qualities (Fig. 6) which included excellent, good and poor. With regard to the quality of matured oocytes , tables (1, 2 & 3) and figure (4) showed the the total number and average number as well as the percentage of the three classes of matured buffalo oocytes per ovary collected from investigated four ovarian groups . The average number of the excellent matured oocytes per ovary was significantly higher (p<0.01) in both non-aged fertile ovarian group 1 and aged fertile ovarian group 3 (0.53+0.19 and 0.43 + 0.19, respectively) than those of the non-aged infertile ovarian group 2 and aged infertile ovarian group 4 (0.03+0.02 and 0.04+0.02, respectively). The average number of the good matured oocytes per ovary was significantly higher (p<0.01) in both of non-aged fertile and aged fertile ovarian groups (0.93+0.16 and 0.62+ 0.08, respectively) than those of non-aged infertile and aged infertile ovarian groups (0.13+0.05 and 0.09 + 0.07, respectively). The average number of the poor matured oocytes per ovary was significantly higher (p<0.01) in each of non-aged infertile ovarian group 2 and aged infertile ovarian group 4 (0.18 + 0.05 and 0.18 + 0.05, respectively) than those of the non-aged fertile ovarian group 1 and aged fertile ovarian group 3 (0.35+0.05 and 0.15 + 0.5, respectively). 2.2-Cleaved oocytes (IVF-Model 1) The average number and proportion of oocytes cleaved to 2 to 4 cell at 48 hours post insemination are given in tables (1, 2 & 3) and figure (4). The results of the present investigation revealed that, the non-aged fertile ovarian group 1 yields a higher number of cleaved oocytes per ovary (P<0.01) than that of aged fertile, non-aged infertile and aged infertile ovarian groups (1.17+ 0.10 vs 0.59+0.08, 0.15+0.09 and 0.16+0.07, respectively). Moreover, when the last three groups were compared, the aged fertile group yield a higher number of cleaved oocytes per ovary (p<0.01) than that of non-aged infertile and aged infertile ovarian groups (0.59+0.08 vs 0.15+0.09 and 0.16 + 0.07, respectively). The aged infertile ovarian group resulted in a non-significant lower yield of cleavage rate than that of non-aged infertile group (0.16 + 0.07 vs 0.15 + 0.19, respectively). 2.3- IVMFC- derived buffalo embryos (IVC-Model 1) The results of IVMFC-derived buffalo embryos of the four ovarian groups indicated that the non-aged fertile group 1 yield a higher number of IVMFC- derived embryos per ovary (p<0.01) than that of aged fertile , non-aged infertile and aged infertile ovarian groups (0.48+0.2 vs 0.12+0.03; 0.04+0.04 and 0.02+0.04, respectively) Moreover, when the last three ovarian groups were compared, the aged fertile group yield a higher number of IVMFC- derived embryos per ovary than that of non-aged infertile (p<0.01) and the aged infertile ovarian group - p<0.05 (0.12+0.03 vs 0.04+0.04 and 0.02+0.04, respectively). However the aged infertile ovarian group resulted in a non-significant lower yield of IVMFC- derived embryos per ovary than that of non-aged infertile (0.04 + 0.04 vs 0.02 + 0.04, respectively). By comparing the results of the four ovarian groups (Tables 1, 2 & 3 and Fig. 3 & 4) for oocytes recovery yield and their developmental competence (IVM / IVF / IVC) .The four ovarian groups were arranged into the following grades: Grade 1 (Very good): the non-aged fertile ovaries having functional structures. Grade 2 (Good): the aged fertile ovaries having functional structures. Grade 3 (Fair): the non-aged infertile ovaries having pathological or no structures. Grade 4 (Poor): the aged infertile ovaries having pathological or no structures. 3- Oocytes developmental competence of non-aged fertile ovarian group1 on Model 1 and Model 2 of IVM / IVF / IVC techniques 3.1- Matured oocytes (IVM) From table (4) and figure (13), the results of the present study revealed that the percentage of matured oocytes for model 1 and model 2 technique was (82.5 % vs 81.5%, respectively) .The differences between the values of both models were non-significant (Table 5). Concerning the quality of matured oocytes model 1 resulted in a higher values of excellent matured oocytes (P<0.01) than that of model 2 (29.9 % vs 19.2 %, respectively). The differences between the values of good quality matured oocytes for both models were non-significant (49.9 % vs 48.1%, respectively). Concerning poor quality of matured oocytes, the present results (Tables 4 & 5) revealed that the values for model 2 were highly significant (P<0.01) when compared with model 1 (32.7% vs 20.3%, respectively). 3.2- Cleaved oocytes (IVF) From table (4) , the results of the present study revealed that the average number of cleaved oocytes / trial and the percentages of the cleaved oocytes for model 1 and model 2 technique were (64.3% vs 48.0%, respectively). The differences between the values of both models were significantly high (p<0.01). 3.3- IVMFC - derived buffalo embryos (IVC) For IVMFC - derived embryos, the present results (Tables 4 & 5) revealed that the values for model 1 were significantly high (p<0.01) when compared with model 2 (41.0% vs 27.2%, respectively). 4 -Cryopreservation results 4.1- Comparison of survival rate and developmental competence between vitrified and slow-freezed mature oocytes The results of the present study (Tables 6 & 7 and Fig. 13)revealed that post thawing percentage of morphologically normal mature oocytes frozen by vitrification technique was significantly higher (p<0.01) than that of slow-freeze.(70.6 % vs 61%, respectively). The developmental competence of post thawing normal mature oocytes revealed that the percentages of cleaved oocytes as well as IVMFC- derived buffalo embryos frozen by vitrification technique were significantly higher (P<0.05) than those frozen by slow freezing technique (33.0%, 10.3% vs 22.2 %, 4.4 % , respectively). 4.2- Comparison between the developmental competence of vitrified and fresh (nonfrozen ) immature and mature oocytes 4.2.1 Vitrified and fresh immature oocytes Statistical analysis revealed the presence of significant differences in the maturation and cleavage rates (P<0.01) in vitrified immature than that of fresh one (49.3 %, 13.3 % vs 81.2 %, 41.8 %, respectively). The differences between the percentages of developed IVMFC-derived embryos for vitrified and fresh oocytes were non significant, although the value of the fresh ones was higher (2.6 % vs 11.3%, respectively). 4. 2.2-Vitrified and fresh mature oocytes The values of the cleavage rate and IVMFC-derived buffalo embryos for vitrified matured oocytes did not differ significantly when compared with those of the fresh ones (10.3% vs 11.5%, respectively). 4.2.3- Comparison between survival rate and developmental competence of vitrified immature and mature buffalo oocytes The results of the present study (Tables 6 & 7 and Fig. 14) revealed that the percentages of post thawing morphologically normal vitrified mature oocytes were significantly higher (p<0.01) than that of vitrified immature oocytes (70.4 % vs 55.4 %, respectively). The values of the cleavage rate and IVMFC-derived embryos for vitrified mature oocytes were significantly higher (p<0.01) when compared with those of vitrified immature oocytes (33%, 10.3% vs 13.3%, 2.6%, respectively). 4.3- Comparison between the survival ratee of vitrified immature and mature buffalo oocytes as well as IVMFC-derived buffalo embryos The differences between the percentages of morphologically normal oocytes for vitrified mature oocytes were significantly higher (p<0.01) than that of vitrified immature oocytes (70.6 % vs 55.5 %, respectively). The survival rate for vitrified IVMFC-derived embryos was non-significantly higher than that for vitrified mature oocytes (79.4% vs 70.6%, respectively). 5 - Cytogentical results 5.1- Chromosomal status of mature buffalo oocytes The metaphase 11 stage of matured buffalo oocytes was revealed as second metaphase stage (M 11): the haploid set of the chromosomes (N = 25 X) is spread out, this result confirm the maturation of buffalo oocytes. 5.2- Chromosomal status of buffalo embryos The Chromosomal status of Egyptian water buffalo embryos (Bubalus-bubalis) had been found to be 50 numbers of chromosomes. These results confirm the fertilization and cleavage process. -

PHD Title

In Vitro Fertilization , Processing and Cryopreservation of buffalo oocytes and embryos

PHD Abstract

Abstract Cairo University Faculty of Veterinary Medicine (Beni – Suef) Department of Theriogenology Name : Saber Mohamed Abd Allah Title : In Vitro Fertilization, Processing and Cryopreservation of buffalo oocytes and embryos. Under supervision of : Prof Dr. Ahmed Gomaa M. Hassan Prof Dr. Mohamed Essam M.El -Nahass Prof Dr.El Sayed Mahmoud M. Abd El- Gawad Determination of the age and fertility status of slaughtered animals as well as the morphological aspects of the ovaries prior to follicle aspiration could be used as an indicator for oocytes recovery yield and their developmental competence . Supplementation of maturation medium with hormones (FSH and LH) for IVM resulted in a significantly high (P<0.01) excellent quality of matured oocytes .The utilization of TALP medium for IVF revealed a significantly high (P<0.01) cleavage rate . Supplementation of culture medium with cumulus cells IVC revealed a highly significant IMFC-derived buffalo embryo production .The vitrified mature oocytes were more tolerant and revealed high developmental competence when compared with the slow freezed matured ones (P < 0.01) .The vitrified IVMFC-derived embryos were more tolerant (i.e: revealed high post survival rates ) than vitrified immature and mature oocytes .The vitrified mature oocytes revealed a significantlly higher (P < 0.01) developmental competence than vitrified immature ones. Chromosomal analysis of matured oocytes and IVMFC - derived buffalo embryos was necessary for assessment of oocyte maturation and embryo cleavage . Key Words : In Vitro Fertilization, Cryopreservation , Buffalo oocytes and embryos.