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22/11/2006 Evaluation of the Quality of Deep-Frozen Semen and Selection of an Effective Method of Goat Sperm Preparation for In Vitro Fertilization By.

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Presentation on theme: "22/11/2006 Evaluation of the Quality of Deep-Frozen Semen and Selection of an Effective Method of Goat Sperm Preparation for In Vitro Fertilization By."— Presentation transcript:

1 22/11/2006 Evaluation of the Quality of Deep-Frozen Semen and Selection of an Effective Method of Goat Sperm Preparation for In Vitro Fertilization By W.P.D.K. FERNANDO

2 Introduction Objectives Semen Quality Analysis Selection of a Sperm Preparation method Results & Discussion Conclusion Acknowledgements End CONTENT

3 INTRODUCTION Production & Productivity of Local Goat Demand for kids, meat & milk HIGH & ESCALATING Supply ? Reasons Lack of good quality breeding materials Selling of breedable or pregnant she goats for meat

4 Cont. INTRODUCTION WHAT IS NEEDED Large amount of better breeding materials Sooner the possible At a lower cost* Potentials Many sustainable farming systems AI & ET Good but limited IN VITRO techniques have a higher potential IVM IVF IVC

5

6 The success of the in vitro techniques depends on… Semen factors Semen quality (motility, abnormalities, live/dead ratio, etc.) fresh or cryopreserved ? how long preserved ? semen of which breed ? Sperm preparation why Sperm preparation ? which method ? Classical sperm swim-up method Percoll gradient method Ficoll medium method Method of centrifugation on a discontinuous density gradient and etc) For Sri Lanka …

7 OBJECTIVES To evaluate whether the quality of deep frozen semen is affected by the breed &/or preserved period To select an effective method of sperm preparation for IVF in goat To facilitate the implementation of IVF technology for breed improvement in local goat.

8 EX:1 EVALUATION OF SEMEN QUALITY 58 Cryopreserved semen straws of four cattle Breeds Friesian Australian Milking Zeebu (FrAMZ) Jersey (Jr) Friesian (Fr) Friesian Sahiwal (FrSw) were selected Classified into 5 age categories according to the semen straw preserved year > 20 years 19 – 15 14 – 10 9 – 5 <5

9 Quality Analysis 1 1.1 Motility Visual detection 1.2 Dead Sperm Percentage Eosin/Nigrosin staining method 1.3 Abnormalities Williams staining method Ex1

10 EX:2 SELECTION OF A SPERM PREPARATION METHOD Two sperm preparation methods were compared Classical Sperm Swim-up Method A Simplified Sperm Swim-up Method

11 Ex2

12 Quality Analysis 2 2.1 Cleanliness of the semen 2.2 Motility Before & after preparation Visual Detection 2.3 Concentration Haemocytometer Ex2

13 RESULTS & DISCUSSION

14 Means in a column with the same letter are not significantly different. Alpha = 0.05 BreedMean Motility Age(yrs)Mean Motility FrAMZ24.50 a >2026.54 a Jy 39.06 a 15-1925.30 a Fr 41.88 a 10-1448.75 a FrSw 44.00 a 5-940.89 a <545.25 a 1.1 Sperm Motility Table 1: Sperm Motility of different breeds and age categories Ex1

15 Breed Mean Death% Age (Yrs) Mean Death% FrAMZ 65.63 a >2064.77 a Jy 54.56 a 15-1969.20 a Fr 56.12 a 10-1448.13 a FrSw 53.60 a 5-956.16 a <546.38 a Table 2: Dead sperm percentages of different breeds and age categories Means in a column with the same letter are not significantly different. Alpha = 0.05 1.2 Dead Sperm Percentage Ex1

16 Correlation of sperm motility and Dead Sperm Percentage A highly significant negative (Pearson) correlation (r = -0.882, P<0.01) between Sperm Motility and Dead Sperm Percentage. Ex1

17 Table 3 - Sperm abnormalities according to breed: Means in a column with the same letter are not significantly different. Alpha = 0.05 Breed Abnormalities HeadMidTail FrAMZ18.1 a 12.1 ab 17.9 a Jy14.9 a 8.4 b 11.1 b Fr18.3 a 13.4 a 10.4 b FrSw17.5 a 14.8 a 8.9 b 1.3 Abnormalities Ex1

18 Table 4 - Sperm abnormalities according to storage time: Means in a column with the same letter are not significantly different. Alpha = 0.05 Age Category Abnormalities HeadMidTail >2016.8 ab 9.5 a 12.7 a 15-1917.2 ab 9.4 a 18.2 b 10-1413.6 b 8.4 a 8.9 a 5-0917.4 ab 14.5 b 11.2 a <520.8 a 16.5 b 11.1 a 1.3 Abnormalities Ex1

19 2.1 Cleanliness of prepared sperms Both methods resulted with slightly particulated semen Method SwmupSswmup Initial motility71 a 72 a Final motility90 b 78 a Means with a same letter are not significantly different. Alpha = 0.05 2.2 Sperm Motility of both preparation methods Ex2

20 Table 5 - Sperm concentration after sperm preparation Method SwmupSswmup Sperm Concentration (x 10 6 ) 4.5 a 7.5 a swmup=swim-up method sswmup=simplified swim-up method Means with the same letter are not significantly different. Alpha = 0.05 Ex2

21 CONCLUSION & SUGGESTIONS There is no effect of storage time and breed on motility and dead sperm percentage but they affect on abnormalities. Other factors affecting motility & Dead Sperm Percentage need to be evaluated The simplified swim up method is as effective as the classical swim up method, but the former is more convenient and more economical. But this selection should be broadened to evaluate the fertilization rate

22 ACKNOWLEDGEMENTS Prof. D.P.S.T.G. Attanayake, Head, Dept. of Biotechnology, FAPMSL is deeply acknowledged for his valuable encouragement & guidance. Mr.D.V.S. de S. Gamage Head, Division of Animal Breeding,VRI Mr. A.S.B.Rathnayake Mr. P.A.B.S. Kumara, Division of Animal Breeding, VRI are also deeply acknowledged for their valuable assistance during the research. Mr.W.K.S. Amarasinghe and the staff Central Artificial Insemination Station (CAIS), Kundasale.

23 Dilip Fernando

24 Introduction Objectives Ex 1 Ex 2 Results & Discussion Conclusion

25 Dilip Fernando

26 back Difference

27 Selling of breedable and sometimes pregnant she goats FOR MEAT A recent study revealed that 62% of total number of goats slaughtered for meat was female while 23% of those was pregnant (Chandrasiri et al. 1999). Poor small-scale farmers sell many of the breedable and pregnant animals when they need money, and usually they tend to sell the best animals, irrespective of their reproductive status.

28 Fresh vs. Cryopreserved semen Much effort requiring & expensive to rear and maintain studs No Maintenance cost of a stud Semen from the studs abroad Semen of dead* studs Storage for longer periods

29 Stats Goat population is approximately 405,000 (2004) GDP = 17.2% AGRI <2% LIVESTOCK <1% GOAT MILK ? Demand? Supply?

30 Dead Sperm Percentage Drop of semen - on a clean and warm glass slide Two drops - eosin nigrosin solution on the semen drop Mixed for 10 sec Still for 50 sec A thin smear on the slide Dry in air. Slide on stage of the microscope Examined under x400 power. sperms counted 100 Pinkish sperms - dead sperms.

31 Head abnormalities

32 Mid piece abnormalities Tail abnormalities

33

34 Abnormalities Semen smear on glass slide Air dried and fixed in flame. Absolute alcohol for 4 min and air dried Washed d. water & with 96% ethanol Stained Carbolfuchsin-eosin solution (williams stain) for 10 min Washed with water Located on the stage of LMC

35 AI & ET vs IVF we should not stay here we need develop more gene technologies spawning here and there thus we thought of a far future IVF cant be used for gene manipulation which will be key in future Immediately dead animals semen/oocytes can be used Even oocytes of new born she goats can be matured and used in IVF


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