Studies on in vitro fertilization in Egyptian buffaloes
Abstract: The present study was carried out to investigate the recovery rates, in maturation, in vitro capacitation of buffalo spermatozoa, in vitro development ( co-culture) and in vivo development of fertilized buffalo ,oocytes.
Exp. 1
723 buffalo ovaries were collected from slaughterhouses classified into
ovarian with CL (341 ovary) and without CL (382 ovary). Oocytes were
aspirated from vesicular follicles with diameter 2-8 mm. Total oocytes were classified as flowing :-
1- oocytes with more than 3 layers of cumulus cells.
2- oocytes with less than 3 layers of cumulus cells.
3- oocytes with expanded cumulus cells.
4- oocytes with cumulus cells and partial denuded.
5- oocytes denuded.
The 1 st and 2nd category were used for in vitro maturation as following:-
oocytes from ovarian with or without CL alone under three types of serum
(Oestrus cow serum, Oestrus buffalo serum and buffalo follicular fluid) with four concentrations ( 5, 10, 15 and 20 % ) per each type of serum.
Exp.2
The optimum concentration of each type of serum was used from exp. 1 plus or minus 2.5 % to study effect of concentration of serum on rate of maturation as following:
ECS (17.5-15 -12.5 %),EBS (7.5-5-2.5%) and FFB (7.5-5-2.5 %).About50
oocytes were used per each concentration.
Exp.3
Semen was collected from five mature buffalo bulls by artificial vaginal
to assessment capicitation rates of spermatozoa, by incubation the sample for (1, 2 and 3 h ) at 37 ºC in water bath with two concentration of heparin (20 and 30 mg /ml) in two media ( Ca + + free HnB.O).
Exp.4
A total of 474 feitilized buffalo oocytes was cultured for 7 days to determined the development rates. 172 fertilized oocytes were cultured with medium alone, 160 fertilized oocytes were co-cultured with granulosa cells (GC) and 142 fertilized oocytres were co-cultured with buffalo oviductal epithelial cells (BOEC).
After culture period 7 days, the recorded development rates from 2 cells stages up to blastocyst stages were recarred.
Exp.5
Buffalo oviduct epithelial cells (BOEC) gave the best results in exp.4. In this expo three concentrations of BOEC (0.75 X 106, 1.5 X 106 and 3 X106 cells/ ml were used to determine their effects on in vitro development of fertilized buffalo oocytes.
Exp.6
After two days postinsemination, fertilizing buffalo oocytes were transferred into doe rabbit oviduct (in vivo) for 5 days to assessment of in vivo development of fertilized buffalo oocytes.
The obtained results could be summar-ized as follows :-
The 1st Experiment:
1- Nearly similar number of follicles and oocytes were obtained on each ovary either with or without CL (3.55 and 2.68- Vs. 3.59 and 2.70/ovary respectively).
2- Results indicated that similar number of different oocytes catcbries as
affected by CL presence on buffalo ovary, but average number of oocytes with> 3 layers the highest count on each ovary .regardless CL presence (2.69).
3- The highest recovery rate (46 %) was for oocytes with >3 layer compact oocytes cumulus cells and lowest recovery rate (3-4 %) for (denuded oocytes in both cases of ovaries with or without CL.
4- The effect of bearing CL on ovaries on in vitro maturation of buffalo oocytes was not significant. Regardless presence of CL, metaphase II stage represented the highest distribution, being more than 40 % in both cases of ovanes.
5- Ocytes with >3 layers showed the highest distribution being nearly similar in both ovaries with or without CL (aoout 61.0 %).
6- Buffalo oocytes with metaphase II stage was significantly (P <0.05) higher with oestrus cow serum than buffalo follicular fluid and oestrus buffalo serum 53.8,33.6 and 31.3 %, repectively.
7 - Concentration of serum affected significantly (P < 0.05) only on oocytes with GV and GVBD, being the lowest for 10 % level, although 10% level showed he highest distribution of ooytes wih metaphase II (44.5%).
Experiment II :
1- There are no significant effect of serum concentration of each type on oocytes at different stage of maturation. The best results were obtained with (OCS, BFF and OBCyS) on oocytes at metaphase II were 58, 50 and 40 % respectively.
Experiment III :
Reacted live sperm (L1) was significantly (P < 0.01) higher and unreacted dead sperm (D2) (P < 0.05) and lower significantly (P < 0.05) in unreacted live sperm (L1) and reacted dead sperm with Ca + + Free than BO medium.
2- Sperm capacitation in vitro improvement significantly (P < 0.05) with supplementation of 30 than 20 g heparin /m1.
3- Incubation of spermatozoa for 3 h _higher significant affected on sperm
capacitation in vitro than 1 and 2 h.
4- The incubation of medium with heparin concentration was insignificant on sperm capacitation, but the highest. distribution of L1 and the lowest distributions of L2 and for Ca + +free medium with 30 g heparin/ m1. The opposite was obtained by BO medium with 20 g heparin /ml.
5- The interaction of medium with incubation times on sperm capacitation was significantly (P < 0.05) only on L2 distribution.
6- The interaction of heparin concentration with incubation time on capacitation sperm was significant on L1, L2 and D2 was insignificant on Dl.
Experiment V :
1- The effect of co-culture on in vitro development were only significant (P < 0.05) on 4-cells and 8-16 cells stage. .While, not significant on 2-cells stage, morula stage and total cleavage rate.
2- Based on maturation rate, the percentages of 2-cells and 4-cells significantly were not affected but, percentage of 8-16 cells and morula stage was highest significantly (P<0.05) for medium with BOEC (16.9%) am the lowest (7.0%) for medium alone.
3- Frequency distribution of 8-16 cells and morula stages was significantly
(P<0.05) the highest for medium with BOEC (28.6 and 22.4 %) and the lowest for that without co-culture, being (15.2 and 13 %). However distribution of 2-cells and 4-cells insignificantly decreased for media with GC (24 and 30 %) or BOEC (20.4 and 28.6 %) compared with medium alone, being (30.4 and 41.3 %).
Experiment VI :
1- The results indicated insignificant effect of concentration of BOEC on cleavage stages of buffalo oocytes, although the highest percentage of 8-16 cells, morula and total cleavage rate with 3x106/ml than 0.75 and 1.5x 106/ml
BOEC.
2- Percentages of cleavage ocytes at different stage and total cleavage rate were not affected significantly by BOEC concentration. The highest percentage of morula stage and total cleavage rate was obtained with co-cultured with 3xl06 /m1 BOEC (17.2 and 62.1 % respectively) as compared to the lower concentrations.
3- Frequency distribution of different stages of cleavage was not affected significantly by BOEC concentration.
Higher frequency distribution of morula (27.8%) for concentration 3xl06 than .75xl06/ml (20.0%) and 1.5xl06/ml (23.5%).
Experiment VII:
1-Recovery rate of embryos transferred in ligated oviductal rabbit in ranged between 44.4 to 61. 3 % with overall mean 54.4 %.
2- Overall cleavage rate of total recovery was 42.6 % with 17.6 % morula, 13.2 % in 8-16 cells, 7.4 % in 4-cells and 4:4% in 2-cells stages.
3- Frequency distribution of embryo development at different stages, the overall mean in morula stage 41.4 %, 8-16 cells 31.0 %,4-cells 17.2 % and 10.3 in 2-cells stages.
Publication year 2004
Pages 155p.
Availability location مكتبة معهد بحوث الانتاج الحيوانى - شارع نادى الصيد- الدقى - الجيزة
Availability number 875
Organization Name
Country Egypt
Department Biotechnolog Research Department
Author(s) from ARC
Agris Categories Animal physiology - Reproduction
AGROVOC
TERMS		 Fertilization. In vitro. In vivo experimentation. Maturation. Ovaries. Spermatozoa. Water buffaloes.
Publication Type PhD Thesis
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