Reproduction & Reproductive Technology Handbook 2025 PDF

PRACTICAL HANDBOOK REPRODUCTION AND REPRODUCTIVE TECHNOLOGY 2025 CHAPTER V SEMEN COLLECTION AND FRESH SEMEN EVALUATION PRACTICUM OBJECTIVES 1.​ Understanding various semen collection methods, including principles, procedures, advantage...

PRACTICAL HANDBOOK REPRODUCTION AND REPRODUCTIVE TECHNOLOGY 2025 CHAPTER V SEMEN COLLECTION AND FRESH SEMEN EVALUATION PRACTICUM OBJECTIVES 1.​ Understanding various semen collection methods, including principles, procedures, advantages, and disadvantages 2.​ Understanding the macroscopic evaluation of fresh semen 3.​ Understanding the microscopic evaluation of fresh semen Semen Collection A.​ The Purpose of Semen Collection Semen collection is a process of collecting sperm from ejaculation, which will later be processed for Artificial Insemination (AI). The purpose of semen collection is to obtain a large volume of semen with good quality so that it can be further processed for artificial insemination. One of the primary requirements for artificial insemination is the provision of high-quality semen. In addition, several factors must be considered, such as the correct semen collection technique and maximum efficiency. In semen collection, many aspects need to be taken into account because sperm are highly sensitive to external factors, such as temperature and exposure to sunlight. B.​ Methods of Semen Collection There are six methods that can be used for semen collection: artificial vagina, electroejaculator, massage of the accessory genital organs, semen collection from the vagina of a female cow after natural mating (post-coitus), teasing, which is the massage of the tertiary genital organ (penis), and Semen Epididymal Recovery (SER) 1.​ Artificial Vagina a.​ Definition Artificial vagina is a device used to collect spermatozoa, designed to resemble the physiological conditions of a natural vagina in terms of shape, temperature, and texture. This simulation makes the bull feel as if he is directly copulating with a female. Department of Reproduction and Obstetrics 1 b.​ Principle The bull is allowed to mount a teaser or dummy cow during ejaculation, after which the penis is guided, and the ejaculate is collected using an artificial vagina that has been conditioned to mimic a natural animal vagina. Semen collection using an artificial vagina is the most effective method for large livestock (cattle, horses, buffalo) as well as small livestock (sheep, goats, and pigs) that are in normal condition (not disabled) and have good libido. c.​ Structure Fig 1. Artificial Vagina No. Gambar Keterangan 1. Outer liner -​ Its length adjusts to the length of the animal's penis. -​ It has an opening and a vent for inserting warm water and air to resemble a natural vagina 2. Inner liner -​ It is longer than an outer liner, with its end folded outward over the end of the outer liner with the help of a rubber band -​ Prevents excessive friction that could cause injury to the penis. 3. Rubber band -​ Locks the outer liner and inner liner to prevent them from coming loose easily. Department of Reproduction and Obstetrics 2  4. Cone -​ Prevents semen from spilling. -​ Connect the sleeve to the collection tube. 5. Collection tube -​ Collects semen. -​ Installed at the end of the cone. 6. Protective cloth for collection tube -​ Protects semen from temperature changes and direct sunlight, as direct sunlight can reduce sperm viability and decrease sperm fertility. d.​ Assembly Instructions ​ Prepare all parts of the artificial vagina ​ Attach the inner liner to the outer liner by folding the end of the inner liner outward and securing it with a rubber band. ​ Attach the cone, which is already connected to the collection tube, to one end of the cylinder ​ Fill the cylinder with water (temperature ±60°C) up to 2/3 of the tube's volume, leaving 1/3 of the volume filled with air ​ Apply sterile lubricant to the entire opening of the artificial vagina e.​ Procedure ​ Preparation ○​ Prepare the holding area with bedding ○​ Prepare a dummy cow or a teaser in squeeze chute ○​ Prepare the bull by cleaning the abdominal area near the prepuce; the preputial area must be washed with running water, cleaned, and dried with a towel ○​ Prepare the artificial vagina, filled with warm water and air, then apply lubricant to the artificial vagina ○​ Prepare a protective cloth to cover the collection tube Department of Reproduction and Obstetrics 3  ​ Implementation ○​ The male is brought to the holding area and directed behind the teaser to increase libido ○​ False mounting (allowing the bull to mount the teaser or dummy cow without ejaculation) is performed 2-3 times ○​ During mounting, the penis is directed into the artificial vagina at a 45° angle ○​ Ejaculation is indicated by a pelvic thrust movement ○​ The artificial vagina is immediately held upright and gently shaken to ensure the semen enters the collection tube, which is covered with a protective cloth. ○​ The artificial vagina is carefully removed to avoid injuring the penis ​ Aftercare ○​ Rinse all equipment with clean water ○​ Wash all equipment with warm soapy water (use a brush) ○​ Rinse all equipment with clean hot water. ○​ Sterilize glassware by boiling for 10 minutes or placing it in an oven (93-148°C) for 30 minutes ○​ Dry all equipment f.​ Things to consider: ​ Adequate facilities are required for semen collection using the artificial vagina method. The bull should be trained, a trained operator should conduct the procedure, and a teaser (substitute for a female cow) must be available. ​ A special pen is needed for the dummy cow or teaser to be mounted. The breeding bull must be properly restrained. A squeeze chute is necessary to protect the handlers and restrain the teaser. ​ The bull must be disease-free, clean, and well-maintained. Before semen collection, the bull's abdomen and preputial area must be washed with running water, cleaned, and dried with a towel. ​ The artificial vagina must be clean g.​ Advantages ​ The semen collected is of higher quality and cleanliness compared to other methods ​ The collected semen volume is relatively high ​ The spermatozoa concentration is sufficiently high ​ Low risk of contamination ​ Allows observation of the bull’s libido level and abnormalities in erection or ejaculation mechanisms h.​ Disadvantages ​ Requires a trained bull and suitable teaser ​ The bull must have high libido Department of Reproduction and Obstetrics 4  ​ Difficult to use on wild or obese bulls ​ Higher risk of injury i.​ Comparison Bull Goat Stallion 2.​ Electroejaculator a.​ Definition A device that delivers a low-power electric current between two electrodes inserted through the rectum to stimulate the accessory glands and ejaculation muscles. b.​ Principle The electroejaculation method is used in livestock when semen collection using an artificial vagina is not feasible. The probe of the electroejaculator is inserted into the rectum and applies a gradual, low-power electrical stimulus to induce erection and ejaculation. The nerves involved in penile erection and ejaculation, namely the pudendal and hemorrhoidal nerves, originate from the lumbosacral plexus, which branches into the pelvic genital organs. Stimulation of nerves in the prostate gland triggers erection, while stimulation of the ampulla and vesicular glands induces ejaculation. Erection is controlled by the parasympathetic nervous system, whereas ejaculation is stimulated by the sympathetic nervous system. c.​ Structure Department of Reproduction and Obstetrics 5  Fig 2. Electroejaculator ​ Probe​ ​ : Conducts electrical impulses to the nerves that control ejaculation ​ Voltmeter​ : Measures and displays the electrical voltage. ​ Battery​​ : Serve as the power source ​ Cable​ ​ : Connects the power supply to the probe. d.​ Procedure Fig 3. Electroejaculator usage procedure ​ Ensure the electrodes on the intrarectal probe are clean ​ Check that the stimulation device is functioning properly ​ Restrain the animal in a squeeze chute ​ Clean the rectum of feces to allow optimal probe function ​ Gently massage the accessory glands for 30 seconds before inserting the probe ​ Apply lubricant gel to the intrarectal probe ​ Move the tail aside to avoid obstruction, then slowly insert the probe ​ Start electrical stimulation by gradually increasing the voltage, holding for 2-3 seconds, then lowering it to 0V (repeat as needed) ​ Observe the animal’s response (each animal may react differently) ​ Stop stimulation once the animal shows signs of impending ejaculation ​ The animal will relax its back, push its hips forward (pelvic thrust), and ejaculate Department of Reproduction and Obstetrics 6  e.​ Things to Consider: ​ Animals urinate immediately after ejaculation, so timing is crucial when removing the semen collection container. Poor timing may lead to urine contamination of the semen. ​ If stimulation reaches 15V with poor response, discontinue the procedure to avoid nerve damage or injury ​ It must target the spinal cord and lumbar region between the 4th lumbar vertebrae and the 1st sacral. If the probe stimulates the wrong vertebrae, erection will not occur, and the ejaculate will be thick. f.​ Advantages ​ Can be performed on animals unable to mount due to paralysis, leg injuries (lameness), or impotence. ​ Suitable for wild animals ​ Produces a higher semen volume than teasing or other manual methods g.​ Disadvantages ​ Semen quality is lower than with the artificial vagina method, with lower sperm concentration ​ Time-consuming and expensive ​ Can cause injury if not performed carefully 3.​ Massage Fig 4. Massage a.​ Definition A method performed by palpating the accessory glands to stimulate semen release. Department of Reproduction and Obstetrics 7  b.​ Principle This technique involves inserting a hand into the rectum and massaging the seminal vesicles and ampulla, followed by stroking the urethra in a caudal direction to induce ejaculation Suitable for large livestock (cattle, buffalo, horses) and poultry (turkeys and chickens).In large livestock, this method is used when a genetically superior male cannot mate naturally due to low libido or leg issues (paralysis, lameness, or injury). For chickens and turkeys, back massage is the most effective semen collection method. c.​ Procedure ​ The male is first stimulated using a teaser or dummy, ensuring no actual mating occurs. ​ Ensure the preputial and rectal areas are clean, as semen drips through the preputium and surrounding hairs, increasing the risk of contamination by dirt and urine. ​ The operator applies lubricant to their hand and gently inserts it into the rectum, then clears feces completely ​ Apply slight downward pressure to locate and massage the seminal vesicles and ampulla toward the caudal direction until ejaculation occurs and the semen is ready to be collected in a collection tube ​ Another assistant is required to collect the semen as it is expelled from the penis or preputial opening. d.​ Advantages ​ It can be used for impotent males ​ Alternative to the artificial vagina and electroejaculator methods ​ Useful for detecting abnormalities in the accessory glands ​ Suitable for males with low libido or those unable to mount. ​ Minimal equipment required e.​ Disadvantages ​ High contamination risk in the semen ​ Low sperm volume and concentration (diluted); mixed with seminal vesicle secretions ​ Low-quality semen ​ May cause trauma to the male 4.​ Teasing Department of Reproduction and Obstetrics 8  Fig 5. Teasing a.​ Definition The teasing method is commonly used for semen collection in dogs. This method involves massaging the bulbus glandis of the dog's penis using the thumb and index finger, stimulating arousal and erection. Once erection occurs, the penis is directed into a collection tube, and the massage continues until ejaculation is achieved. The stimulation process can be assisted by the presence of a female dog in estrus. b.​ Principle Massage of external organs until the animal achieves erection c.​ Advantages ​ Effective for dogs ​ Quick and easy ​ Simple, does not require additional equipment d.​ Disadvantages ​ Easily contaminated by mucus and urine ​ May cause penile trauma 5.​ Post-coitus Fig 6. Post-coitus Department of Reproduction and Obstetrics 9  a.​ Definition Post-coitus is a semen collection method performed immediately after natural mating. This method utilizes a pipette and a long spoon. It is not recommended as the semen is contaminated with mucus and urine, increasing the risk of genital disease transmission. b.​ Principle After copulation, semen is collected from the anterior part of the vagina using a long sterile spoon. It can also be obtained from semen droplets that exit. c.​ Advantages ​ Quick and easy ​ Simple, requires minimal equipment ​ Performed without coercion d.​ Disadvantages ​ Easily contaminated by bacteria during collection ​ Contaminated with female reproductive tract secretions ​ Requires natural mating beforehand 6.​ Semen Epididymal Recovery (SER) a.​ Definition SER is a semen collection method through the epididymis. This method collects semen from the cauda epididymis and the proximal ductus deferens. The SER method is considered valuable as it can be performed on recently deceased males or living animals with erectile dysfunction or sexual disorders. For living animals, SER is conducted after castration. This method has been reported to be applicable to domestic animals, laboratory animals, and wildlife. Semen collection using SER is performed when natural mating is not possible due to difficulties in handling aggressive animals or sudden death. This method is applied to animals in life-threatening conditions where spermatozoa are still present in the epididymis. b.​ Methods and Principle SER is performed by directly collecting semen from the cauda epididymis. Several techniques can be used for post-mortem semen collection using the SER method, including: ​ Incision Method → This involves making multiple incisions on the cauda epididymis using a blade until seminal fluid is released. Several incisions are made in the cauda epididymis to allow spermatozoa to flow out and be collected. Department of Reproduction and Obstetrics 10  Figure 7. Incision method ​ Aspiration Method → An incision is made on the epididymis, and sperm is aspirated using a pipette for collection.. Figure 8. Aspiration method ​ Float up Method → The cauda epididymis and the proximal part of the ductus deferens are incised and placed in a petri dish containing semen extender medium.. Figure 9. Float up method ​ Retrograde Flushing Method → A cannula or syringe is inserted into the ductus deferens, and semen extender is flushed in a reverse flow from the ductus deferens to the cauda epididymis Department of Reproduction and Obstetrics 11  Figure 10. Retrograde flushing method c.​ Advantages ​ High semen quality as it is collected directly from the storage site (epydidimis) ​ A large number of spermatozoa can be obtained ​ Applicable for animals that die suddenly or for endangered species to preserve genetic lineage ​ The collected spermatozoa can be used to produce multiple straws for artificial insemination or IVF d.​ Disadvantages ​ Prone to contamination by blood or surrounding tissues. ​ Semen quality may degrade if not properly packaged or if tissue degeneration occurs ​ Sperm must be collected and cryopreserved as soon as possible to maintain semen quality ​ Requires a considerable amount of equipment Department of Reproduction and Obstetrics 12  Fresh Semen Evaluation Semen quality tests are carried out immediately after collection or before dilution. These tests include macroscopic examination (volume, color, consistency, pH) and microscopic examination (mass motility, individual motility, percentage of live and dead sperm, concentration, and abnormalities). A.​ Macroscopic Evaluation 1.​ Volume Semen volume examination is conducted directly using a conical tube. Bull and ram semen have a small volume but a high sperm concentration, while stallion and boar semen have a larger volume but a lower sperm concentration. Factors that affect the volume of semen ejaculated include the age of the male, physical condition, health status, environment, season, skill, nutrition, semen collection procedures, frequency of collection, breed, and others. Semen volume is not the main factor in the fertilization process, but the factor that determines the success of fertilization is the total number of spermatozoa per ejaculation. Semen volume in several species is as follows : ​ Bull​ ​ : 5 – 8 cc ​ Ram​ ​ : 0,8 – 1,2 cc ​ Boar​ ​ : 150 – 200 cc ​ Stallion​ ​ : 60 – 100 cc ​ Dog​ ​ : 3 – 80 cc (ejaculation in dogs occurs 3 times) ​ Rooster​ : 0,2 – 0,5 cc 2.​ Color Normal semen generally has a milky or whitish-cream color and is cloudy. The degree of cloudiness in semen depends on the concentration of sperm cells (the cloudier it is, the higher the concentration of sperm cells). a.​ Normal semen color in several species is as follows : ​ Bull​ ​ : slightly creamy white ​ Ram​ ​ : creamy white ​ Boar, Stallion​ : cloudy grayish white ​ Rooster​ : milky white b.​ Color abnormality No Color Abnormality Description 1. Yellowish The presence of riboflavin pigment Department of Reproduction and Obstetrics 13  2. Yellowish green The presence of Pseudomonas aeruginosa bacteria. 3. Dark red to pink The presence of blood coming from the penis or urethra. 4. Light brown or greenish There is a possibility of contamination with feces. 5. Brownish (dark brown) There is blood decomposition. 6. Clumps, clots, and fragments in The presence of pus, which generally the semen. originates from the accessory glands. 7. Gray The presence of contamination with dirt or debris, such as smegma (skin secretions, desquamation of epithelial cells). 3.​ Consistency Semen consistency or viscosity testing can be done by gently shaking the tube containing the semen. Semen consistency is related to the color of the semen, as knowing the normal color can help predict the spermatozoa concentration. The cloudier the semen, the higher the concentration. Grade Consistency Concentration (million cells/ml) 1 Watery 2000 4.​ Degree of acidity (pH) The degree of acidity greatly affects the survival of spermatozoa. pH testing is performed using pH strip test, litmus paper, or a pH meter. Sperm metabolism in an anaerobic state produces lactic acid, which accumulates and increases the acidity (lowers the pH) of the solution. pH can also be affected by concentration; when the concentration is high → more spermatozoa → higher sperm metabolism → increased lactic acid → more acidic (lower pH). Department of Reproduction and Obstetrics 14  The normal pH of semen in several species is as follows: : 1.​ Bull​ ​ : 6,4 – 7,8 2.​ Ram​ ​ : 5,9 – 7,3 3.​ Boar​ ​ : 7,3 – 7,8 4.​ Stallion​ : 7,2 – 7,8 5.​ Dog​ ​ : 6,3 – 6,7 6.​ Rooster​ : 7,2 – 7,6 5.​ Odor Normal semen generally has a characteristic fishy odor, accompanied by the odor of the animal itself. A foul odor is typically found in semen that contains pus due to an infection in the reproductive tract. B.​ Microscopic Evaluation 1.​ Mass Movement Mass movement is examined at body temperature by placing a drop of semen on a object glass that has been warmed, without a deck glass and without dilution. It is then observed under a microscope with low magnification (around 10 x 10). Mass movement evaluation can only be performed on fresh semen ejaculates. Motility and mass movement assessments are very sensitive to temperature. Low temperatures can inhibit motility, while high temperatures can increase motility. Temperatures should be kept below 50°C, as sperm will die quickly above this temperature. a.​ Criteria for Mass Movement Evaluation 0 poor​ ​ : No or very little individual movement (+) fair​ ​ : No waves, but active progressive individual movement (++) good​​ : Thin, small, infrequent, unclear waves, moving slowly (+++) very good​ : Large, numerous, dark, thick waves, active, like fast-moving black cloud clumps. 2.​ Individual Motility Motility is the percentage of spermatozoa that move straight forward in one field of view (under a microscope). Individual motility can be influenced by endogenous factors (such as sperm age, spermatozoa maturation level, biochemical properties, and factors that affect energy availability) and exogenous factors (such as viscosity, pH, temperature, and ion composition in the surrounding media). Individual motility assessment can be performed on fresh semen or frozen semen that has been diluted. To determine individual motility, semen is placed on a object glass and then observed under a microscope with 40x magnification at body temperature, covered with a deck glass. a.​ Types of spermatozoa movement ​ Progressive​ ​ : Sperm movement moving straight forward Department of Reproduction and Obstetrics 15  ​ Circular ​ ​ : Sperm movement in a circular motion, usually caused by cold shock ​ Reverse/retreat​ : Sperm movement moving backward, usually caused by cold shock ​ Vibratory​ ​ : Sperm movement vibrating in place ​ Oscillatory ​ ​ : Sperm movement swinging, usually in older sperm ​ Imotile/necrospermia : Sperm does not move Spermatozoa motility is considered normal when they exhibit progressive movement. A&B​ : Progressive ​ C​​ : Oscillatory D​​ : Circular b.​ b.​ Individual motility assessment scores ​ 0 : Sperm does not move ​ 1 : 0–30% progressive, circular movement in place ​ 2 : 30–50% progressive, swinging or circular movement ​ 3 : 50–80% progressive ​ 4 : 80–90% progressive ​ 5 : 100% progressive Spermatozoa motility is considered good when they exhibit progressive movement with a percentage of 70–100%. c.​ Spermatozoa Motility Examination Steps : 1.​ Prepare physiological NaCl at a temperature of 37°C. 2.​ Make a semen and NaCl mixture with a 1:4 ratio. 3.​ Drop the semen and NaCl mixture onto a warmed object glass, then cover it with a deck glass.. 4.​ Next, examine it under a microscope with 40×10 magnification, observing at least 3 fields of view. 5.​ Observe the spermatozoa movement and calculate the percentage of sperm that are progressive, meaning they move forward. Department of Reproduction and Obstetrics 16 3.​ Spermatozoa Concentration Based on Head-to-Head Distance Spermatozoa concentration refers to the number of spermatozoa per milliliter of semen. The concentration of spermatozoa can be measured using a hemocytometer, colorimeter, or spectrophotometer. Sperm cell concentration can be calculated by assessing the head-to-head distance between sperm cells. Simply, the semen is diluted 200 times with water or formalin, and then observed under a microscope with 400× magnification. The results can be classified as follows : a.​ Densum (D) or dense: The distance between the two closest sperm heads is less than the length of one head. The spermatozoa concentration is estimated to be 1000–2000 million cells/ml of semen. b.​ Semidensum (SD) or moderate: The distance between the two closest sperm heads is equal to 1–1.5 times the length of the sperm head. The spermatozoa concentration is approximately 500–1000 million cells/ml of semen. c.​ Rarum (R) or rare: The distance between the two closest sperm heads exceeds the length of one sperm head or is equal to the length of the entire spermatozoon. The spermatozoa concentration is 200–500 million cells/ml of semen. d.​ Oligospermia (OS) or few spermatozoa: The distance between the two closest sperm heads exceeds the length of one spermatozoon. The concentration is