Lecture 2. semem collection lecture 2 & 3

self prepared lecture

1. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 1 31/03/2017

2. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 2 Semen Collection And Evaluation

3. Learning Outcomes; At the end of this lesson, each one of you should be able to; List down methods of semen collection Identify parts of AV Remember the Advantages and disadvantages of AV, normal values of the bull sperm, freezing methods and packaging methods. Explain the reasons of semen extension Recall the advantages and disadvantages of frozen semen. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 3

4. METHODS OF SEMEN COLLECTION LEARNING OUTCOMES . After the completion of this lesson, each student should be able to; R ecall various methods of semen collection. Describe advantages and disadvantages of each method. Explain semen collection by AV method and precautions. Demonstrate the procedure to prepare artificial Vagina. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 4

5. Preparatory Activities For Semen Collection Semen collection is like harvesting any other farm crop. Effective harvesting of the semen involves obtaining the maximum number of sperms of highest possible quality in each ejaculate. The ultimate objective is to make maximum use of superior sires . Semen collection is a complex procedure involving coordinated efforts between the animal handler and the collector. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 5

6. BULL PREPARATION Why is it essential? • To decrease microbial load • To increase semen volume, sperm concentration • To decrease reaction time PROCEDURE • Prepucial hairs must be clipped leaving a tuft of 2 cm all around. •Prepucial washing with warm normal saline may be used to decreases microbial load. •Clean and wash the bull, especially the hind quarter 1 hour before the semen collection. •Ordinary washing soap and mild brush may be used. •Special attention must be given on lower abdomen area and prepuce. •Paper towel or damp towel may be used to remove excess water 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 6

7. 2. TEASER / DUMMY PREPARATION Teaser may be a male or a female in estrus. It is also cleaned the same way as for bull. Special attention should be given to the back and perineum area. 3. WASHING AREA Washing area should not be more than 20 m away from collection area / serving area. It should be made of rough concrete. Floor should be slanting with drainage facility of water, dung etc. Adequate facility of water with reasonable pressure must be there. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 7

8. 4. COLLECTION AREA It should be away from noisy places. It should not be more than 30 m away from semen evaluation lab. The ground must not be slippery / muddy. Collection area should be fenced with strong metallic bars to avoid escape of bull. There should be one slanting metal stanchion fitted with wooden arms for the restraint of teaser. Sufficient space should be there for free movement of bulls and the semen collector. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 8

9. Methods of semen collection 1.Pan collection method 2.Vaginal spoon method 3.Sponge method 4.Breeder’s bag method 5.Sperm collector method 6.Artificial vagina (AV) method (most commonly used) 7.Massage method (Used for semen collection from lame bulls and in dogs) 8.Electro ejaculation (From lame bulls) 9.Fistula method 10.Dummy females 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 9

10. SEMEN COLLECTION BY AV METHOD The AV Method of collecting semen is the fastest and most sanitary of the various methods available. It has been proved to be the best method and is now most commonly used. WHY IS IT DONE? Semen is collected in natural status Semen is collected in hygienic condition Frequency of semen collection: It should be done 2-3 times a week. More frequent semen collection results in poor semen quality. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 10

11. Artificial Vagina Assembly 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 11

12. ADVANTAGES Practically the whole ejaculate is collected in uncontaminated and natural stage. It is free from the extraneous secretions. Sterile conditions of the apparatus ensure disease control. The viability of the sperm is better. No female is needed if dummy is a success. DISADVANTAGES Occasionally it is difficult to get the males to serve the artificial vagina. The apparatus involved is slightly costly and requires technical hands. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 12

13. SEMEN EVALUATION WHY IS IT ESSENTIAL? To investigate the infertility problems For breeding soundness evaluation of bulls For semen extension and processing 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 13

14. METHODS OF SEMEN EVALUATION A.Physical / macroscopic Color :creamy to milky white color Volume: Consistency: pH:6.6 to 6.9 B.Microscopic Mass motility Individual motility Sperm concentration Live sperm count Abnormal sperm count C. Biochemical Fructolysis index Methylene blue reduction test Resazurin reduction test Rating Grade Type of wave motion +++++ Good Very dense fast wave motions just like sea waves ++++ Fairly good Waves are comparatively not so rapid as above +++ Average Slow and thin waves ++ Poor Waves absent, progressive individual movement visible ++ Unsatisfactory Only about 20% may show progressive scattered movements Mass Activity 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 14

15. Parameters Cows Color creamy Volume 3-5 ml Consistency Rough estimate of sperm concentration Concentration 1000-1500 millions/ml Motility >50% with forward progression Live sperm >50% Total sperm abnormality <20% Normal Values of Semen 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 15

16. Normal Sperm Morphology 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 16

17. Sperm Abnormalities Rationale: Only morphologically normal sperm participate in fertilization process which is prerequisite to fertilization. Sperm abnormality is detected by Nigrosin staining technique ( Practical Class). Many abnormalities of structure may occur ; •In the development of spermatozoa •In the handling: → during handling → during collection valuable diagnostic and in assessing the potential fertility of bulls. These may be in the head, middle piece or tail, which may interfere with progressive motility and fertility of spermatozoa. Classified as; 1.Primary : caused by aberration during spermatogenesis. 2.Secondary: aberrations at the subsequent developmental stages i.e. after spermatogenesis is complete and sperm has left the seminiferous tubules. Usually due to dysfunction of epididymis, seminal vesicles and genital diseases. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 17

18. Primary Sperm abnormalities brought about during spermatogenesis may include in; Head : mega, micro, pyriform elongated, double, and abnormal, detached . 2.Middle piece: abaxial attachment, thick short and 3.Tail : double , stumpy, ring form Primary sperm Abnormalities Normal Decapitated Macro cephalic Micro cephalic & Stump Tail Pyriform Round and Double Tail Pyriform Round head micro cephalic 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 18

19. Secondary abnormalities could be due to dysfunction of epididymis, seminal vesicles and genital diseases. include normal detached heads, loose heads, detached, coiled mid piece, presence of cytoplasmic droplet. Secondary sperm Abnormalities Bent Tail Coiled Tail Folded tail Double Tail 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 19

20. Semen extension to extend its volume so that a large number of females can be inseminated from one ejaculate having minimum number of spermatozoa to achieve fertilization. It is the dilution of semen with nutrients to increase the life of sperm and volume of semen. Basic components of an extender 1.Egg yolk : Membrane stabilizer, cryoprotectant 2.Buffer : Provides buffering capacity to semen 3.Antibiotics : To check microbial growth Chilled semen the extenders used are : •Egg yolk-citrate extender ………………..: Cow bull and buffalo bull • Tris -yolk extender……………: Buffalo bull 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 20

21. For DFS the extenders used are :-196 • Egg yolk-citrate-glycerol extender : Cow and buffalo bull • Tris-yolk-glycerol extender : Buffalo bull Antibiotics added to the extender • Penicillin – 1000 IU/ml • Streptomycin – 1000 μg /ml Composition of citrate buffer; Sodium citrate dihydrate :2.9 g Penicillin G sodium :1 lac Unit Dihydrostreptomycin sulphate :100 mg DDW :100 ml Composition of Tris buffer; Tris buffer :3.028 g Citric acid monohydrate :1.675 g Fructose (Anhydrous) :1.250 g Penicillin G sodium :1 lac Unit Dihydrostreptomycin sulphate :100 mg DDW :100 ml Buffer is autoclaved and stored at 4ºC 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 21

22.  Basic qualities of an ideal extender: Should maintain osmotic pressure Should maintain PH Should have Buffering capacity Should provide nutrition Should protect sperm against cold shock Non-toxic , easy to prepare and easily available Compatible with a antibiotics Should permit clear sperm picture under microscope. Should not render cleaning of glassware or semen containers difficult .  Basic contents of an extender : Egg yolk – membrane stabilizer, cryoprotectant Buffer – buffering capacity to semen Antibiotics – check microbial growth 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 22

23. Semen Preservation or Methods of Freezing Refrigeration Method at 4-6 ° C Ultra low temperature at bovine sperm frozen at ־ 79C temp. in dry ice or Liquid Nitrogen at -196 degree. semen at room temp at 18-25 degree Celsius. Advantages of frozen semen:  Selective mating.  Early progeny testing.  Maximum utilization of sires.  Long storage.  Reduced transportation cost.  Disease control.  Feeding and management cost of bulls reduced.  Progeny even after death of the bull.  No wastage of semen.  No need of sterilization of AI gun.  No fear of breakage of AI gun. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 23

24. Disadvantages of frozen semen Semen from about 10 to 20 per cent of bulls will not withstand freezing. These are often bulls of low fertility . The ampouling , freezing and storage equipment are very costly . In the freezing process about 40 per cent spermatozoa are killed. So increased number of spermatozoa per insemination are required . If proper bull health is not maintained, frozen semen has great potential for the spread of viral and bacterial diseases . 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 24

25. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 25 Packing Methods For Freezing The straw method was introduced by scientist of Denmark during 1940 for packing of liquid semen . Adler (1960) first frozen the semen packed in straws by using liquid nitrogen vapor. This technique was later modified by Cassou in 1965 which was called as “medium straws”. This was in use for a period of time. Later Cassou in 1968 brought further improvement by reducing diameter for better freezing which is called as “mini straws”. Most of the straws recently used were called as “French straws” - made of poly vinyl chloride.

26. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 26 Glass ampoules (Van Demark and Kinney, 1954 ). German straws (Simmet, 1972- Mini tubes-Landshut method – 65 mm x 2.8 mm – 0.25 ml capacity) S.No. Type of straw Length Diameter Volume 1 French medium 135 mm 2.8 mm 0.5 ml 2 French mini 135 mm 2.0 mm 0.25 ml 3 German straw 65 mm 2.8 mm 0.25 ml The dimensions of French and German straws are as follows  Freezing technique: French (Cassou) technique is used in these days

27. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 27 The French straws have two ends – factory seal end and laboratory seal end. In this the factory seal end was formed in the manufacturers themselves . The laboratory seal end will be open when it is purchased. The laboratory seal has to be created in the semen processing laboratory after filling the straw with diluted semen before freezing. Factory seal has two cotton plugs with PVA powder in-between them. This seal is formed by while manufacturing semen straw at factory. Laboratory seal - formed with PVA powder or sealing machine after filling of semen. This seal is formed at semen processing laboratory.

28. Thawing of frozen semen: Warm water (35 - 40C temp.) 30 sec. is the best. Storage and shipment of semen: Once semen is stored at lower temp., there should never be change in the temp. of stored semen. Semen should also be transferred to other places at the same temp. at which it is stored. The vial containing chilled semen should be wrapped with paper before it is placed in thermos having ice, to avoid cold shock to sperms coming directly in contact with the ice in the thermos. Frozen semen should always be transported in the liquid nitrogen containers. 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 28

29. Video ( Semen collection to AI) 04-Apr-17 ANS203 SEMEN COLLECTION & EVALUATION 29