Semen Collection and examination.pptx

bull-semen-AI

1. Semen Collection, evaluation and preservation

2. Methods of semen collection 1. Artificial vagina-cattle, horse 2. Electro-ejaculator - Shaots , wild animals 3. Gloved hand masturbation-dogs, birds, rabbits, etc 4. Post- mortum semen collection- from epididymis in all spp.

3. Artificial vagina Semen collection from bulls using an AV requires three people: one to handle the teaser animal, one to control the bull and one to collect the semen. It is important that the collection area have non-slip flooring to avoid injuries and because ejaculation may be inhibited if the bull is nervous about his footing.

4. Cont… Bulls are heavy and should have regular hoof care. Poor hoof condition can inhibit the bull from mounting or cause pain when dismounting. A steer is most commonly used as a teaser and mount animal. Female teasers are not recommended because of the potential risk of intromission and spreading of venereal disease .

5. Cont… The teaser should be a calm animal of the appropriate size for the bull being collected. The back and rear quarters of the teaser are washed with a disinfectant every collection day. It is also common for the rear quarters to be clipped routinely.

6. Cont… The artificial vagina uses thermal and mechanical stimulation to stimulate ejaculation. The liner of the AV is filled with water at 42-48 degrees Celsius, and the inner surface is lubricated with something like K-Y jelly . An insulating cone is placed over the end from which the collection tube protrudes to avoid subjecting the semen to temperature shock.

7. Cont… The bull's preputial hair should be clipped in preparation for using the AV. These sanitary precautions are intended to minimize microbial contamination of the semen being collected. For collection, the teaser is positioned straight in front of the bull for mounting. Oftentimes, it helps to arouse the bull if the steer is led around the collection arena with the bull behind, then stopped abruptly, similar to the behavior of a cow in estrus.

8. Cont… False mounting is an effective way to sexually stimulate the bull. Providing two false mounts with two minutes of active restraint and one additional false mount maximizes sperm cell numbers. Final preparations are made to the AV between the second and third false mounts. Proper construction of the AV is important to avoid damaging the bull's penis and to avoid stressing sperm cells .

9. Cont… Successful semen collection with an AV depends on the bull being comfortable around people , and they need to be trained to use the AV. However, bulls are big and dangerous, and personnel safety should be emphasized constantly. Most bulls in artificial insemination centers have a nose ring installed as a valuable and humane means of physical control. In artificial insemination centers, bulls are typically collected 2 or 3 times per week, with 2 or 3 ejaculates per collection day. The following images depict routine semen collection from a bull.

10. Electro-ejaculation Electro ejaculators are designed to stimulate the pelvic sympathetic and parasympathetic nerves with pulses of low voltage and amperage to induce penile erection and ejaculation. An electro ejaculator set has the following components: carrying case, rectal probe, control unit, battery charger, power cord, probe cord, semen collection handle, collection cone and a collection vial. It can be used in all male except in stallions , Because of the significant skeletal muscle contractions it causes, electro ejaculation is not used in stallions; except in rare cases, under general anesthesia .

11. Cont… There are different kinds of electro ejaculators available in the market. They may be battery operated or plugged into an electrical outlet. They may be manually operated, operate from a built in program and may be programmable; some have all three options.

12. Cont… Most bulls ejaculate with electrical stimuli of < 9 volts . There are also different kinds of rectal probes that vary in diameter, weight, orientation of electrodes and in the number of electrodes. All modern probes have three ventrally oriented electrodes. Most probes have a U-shaped extension that fits around the bull's tail when the probe is inside the rectum.

13. Cont… The bull’s tail keeps the probe oriented correctly. To obtain the best results with each individual bull it is important to choose the right probe size since its weight and diameter influence transmission of the stimulus to the nerves. Probes with larger diameter produce a stronger response to stimuli of a given electrical output than smaller diameter probes.

14. Cont… The recommended probe diameter for bulls weighing 1200 - 2000 lbs (550 - 900 kg) is 6.5 to 7.5 cm. For larger bulls, a 9 cm diameter probe may be necessary to achieve ejaculation. Bull Restraint Ideally, bulls are restrained in a chute with good footing without the head caught in a head gate. If they are caught by the head, they are more likely to lie down during the procedure.

15. Cont… In rare instances, it may be necessary to prevent a bull from lying down during electro ejaculation by applying a restraining belt under the bull’s chest. A 30 inch (76 cm) wide chute can accommodate most bulls. It is extremely important to locate at least one strong pole behind the bull. With only one restraining pole, a height of 28 - 30 inches (71 - 76 cm) may be preferred if the scrotum and testicles are to be examined.

16. Cont… Technique 1. Move the bull to the chute and position the pole behind. Set up the electro ejaculator beside the bull leaving it turned on and ready to be used. 2. Using a palpation sleeve, the rectum is emptied of feces and a longitudinal massage is applied over the ampullae and urethralis muscle for 1 - 2 minutes.

17. Cont… 3. A lubricated probe is introduced into the rectum with the electrodes facing ventrally. Make sure the electro ejaculator is turned on before performing this step. If it is turned on after inserting the rectal probe, the bull may receive a strong electrical pulse that will increase the level of stress in the animal. 4. Electrical stimulation is begun slowly until the bull shows a minimal response. Consecutive stimuli are then given, each increasing in intensity a small amount. Stimuli should last 1 - 2 seconds and then be discontinued for 0.5 - 1 second before the next one starts.

18. Cont… 5. After several stimulations, clear pre-seminal fluid begins to flow from the protruded penis. This clear pre-seminal fraction should not be collected. 6. As soon as the cloudy sperm rich fraction begins to flow from the penis, a collection cone with the test tube is placed over the penis and the sample is collected. 7. After collecting a suitable sample, the stimulation is stopped and the rectal probe is removed.

19. Hand Massage :   applying pressure and massage to the penis. Or rectal stimulation of Seminal vesicles and Vas deferens. Used for dog and pig Post- mortum semen collection - the semen directly collected from epididymis in all spp.

20. Semen examination Semen samples vary from individual to individual and from ejaculate to ejaculate These variable parameters are:- Volume - Each spp. of domestic animals have normal volume range Ex, Cattle = 10 ml Horse = 75 ml Pig = 200 ml Sheep/goat = 1 ml

21. Cont… 2. Contamination/color of semen Normal color for spp. Ex, Cattle: creamy white/milky Horse: yellowish white Abnormal constituents/contaminants → blood, pus, tissue debris, etc Examination should be done both macroscopically and microscopically

22. Cont… 3. Sperm motility Motility of sperm is evaluated in two ways A. Mass motility – Examine the edge of a drop of semen under the microscope and score swirling movement of semen under low magnification. Ex, 0 = No visible movement 1 = Slight shimmering (tail moving but no progressive movement)

23. Cont… 2 = Slow swirling bands of high opacity and low amplitude (poor progressive movement) 3 = Moderate swirling bands of high opacity (moderate progressive movement) 4 = Rapidly swirling bands of high opacity and high amplitude (excellent progressive movement)

24. Cont… Other methods include:- B. Individual sperm motility : Dilute the sperms and estimate the proportion of sperms that progressively move across the field of vision under medium objective/magnification. Sperms that quiver on a single spot are not moving → this shows B rownian motion.

25. Cont… Determination of Proportion of motile sperm Here the proportion of immotile sperms give a rough estimate of the proportion dead sperms. The incidence of dead sperms low in fresh semen May rise after prolonged sexual abstinence Higher in semen of males with lower fertility

26. Cont… Viability test : An estimation of proportion of dead sperm is made using vital stains Negrosin-oesin staining technique - Oesin stains only dead sperms - Negrosin provides a dark and suitable background to view pale and pink sperms. Dead sperm take the stain and appeared red and live sperm appeared white.

27. Cont… Assessment of abnormal sperm cells Examine the negrosin-oesin smears and count the number of morphologically atypical sperms in a total of 100 sperms Sperms have a characteristics form and size Variation from typical pattern affecting all parts may occur This severely reduces fertility. Some atypical sperms maybe active and not detectable on sperm motility test

28. Cont… Other methods include :- Assessment of acrosomal damage Make a smear of diluted semen Air dry the smear Fix in buffered formolin Geimsa stain for 90 minutes Examine under 100 objective lense

29. Semen extension → dilution of semen to certain concentration or cells/ml It serves to reduce the cell concentration to workable values → a number of inseminations from a single ejaculate A semen diluents or extender is also necessary to for semen preservation

30. Cont… A semen extender:- Provides an energy substrate Stabilizes pH Provides osmotically controlled environment (buffering) Protects against cooling damage

31. Cont… Extenders (7 components) Nutrients Glucose, fructose Cold shock prevention Milk, skim-milk, egg yolk Buffer Citrate, Tris Osmotic pressure The buffer componentInhibit bacterial growth antibiotics Increase volume fluids Cryoprotectant glycerol

32. Semen Cryopreservation After dilution at ambient temperature semen should be cooled over 1-2 hours to 0-5 o C Glycerol may be added at ambient of at 5 o C Equilibration of sperm with glycerol holding time leads to membrane modification for optimum cryo - survival ( cryopreservetion )

33. Cont… Semen packing 1 . Pellet method:- Small drop of diluted semen is frozen on the surface of liquid CO 2 (dry ice). Cooling rate is 20-30 o C/min → maximum 100 o C and pellet volume is 0.3-0.8 ml. G ood for pigs. 2. Straw method:- Use of plastic (polyvinyl) straws with a length of 133 mm and internal diameter of 2.5 mm (0.5 ml) or 1.7 mm (0.25ml). Yields a maximum cooling rate of 170 o C/min

34. Cont… Freezing rate:- For peak survival cool at the rate of 10-60 o C/min. At the rate of 50 o C is good. The critical range of temperature is from 0 o C to -45 o C Below -45 o C cooling can be more rapid and it is possible to plunge the straws into the l iquid N 2 at -50 o C instead of N 2 vapor .

35. Cont… Storage:- Straws and pellets should remain in the liquid N 2 at -196 o C . Small sample volume and large surface area lead to rapid change in temperature. Thawing Straws and pellets are thawed by immersing them into water bath at 37 o C for few seconds before use for insemination.