Veterinary · Clinical · also MSVE VGO-511
Male reproduction and fertility, semen collection, evaluation and freezing, artificial insemination, embryo transfer, IVF, cloning and sexed semen, and reproduction in the non-bovine species.
In the exams: ICAR AICE-JRF core group 07; ICAR AIEEA-PG 13 (ARGO); RPSC (ETT, IVF, cloning)
Andrology is the study of male reproduction and fertility; assisted reproductive techniques (ART) are the laboratory and field methods that multiply the genetic value of the best males and females: artificial insemination (AI), semen freezing, multiple ovulation and embryo transfer (MOET), ovum pick-up with in-vitro embryo production (OPU-IVP), sexed semen and cloning. Together they are why one elite bull can sire tens of thousands of calves on several continents, why a sport mare can compete while carrying no pregnancy of her own, and why a dairy farmer can choose to have a heifer calf.
The subject matters to every practitioner, not only to the semen-station veterinarian. A breeding soundness examination (BSE) before the breeding season is one of the highest-return procedures in beef practice; a bull, ram, boar or stallion that is subfertile damages many females at once, whereas a subfertile female affects only herself. Field AI is the commonest procedure performed by government veterinarians and AI technicians in South Asia and much of Africa, and its success depends on correct semen handling, timing and hygiene more than on any drug.
Examiners treat the subject in two layers. Recall papers (state public-service commissions, ICAR AIEEA-PG, Nepal NLEV, China's national practising examination) ask species values: ejaculate volume and concentration, accessory glands, duration of spermatogenesis, the site of semen deposition, the timing of AI and the history of key techniques. Licensing and PhD papers (NAVLE, RCVS, ICAR AICE-JRF core group 07, specialist boards) ask applied reasoning: classify a bull from his BSE findings, calculate how many doses an ejaculate yields, explain why boar semen is kept at 16 °C rather than 5 °C, or choose between MOET and OPU-IVP for a given donor. This manual covers both. Values quoted are textbook ranges; where they differ by breed, country or laboratory, the text says so. Any drug mentioned is named by class or active ingredient only, and any dose — confirm against the product label and your national formulary.
The male tract has four parts: the testes, which make spermatozoa and testosterone; the excurrent ducts (efferent ducts, epididymis and ductus deferens), which mature, store and transport sperm; the accessory sex glands, which add the seminal plasma; and the penis with its prepuce. The testis is covered by the tunica albuginea and divided into lobules of seminiferous tubules. Inside the tubules, Sertoli cells support developing germ cells, form the blood-testis barrier with their tight junctions, secrete androgen-binding protein and inhibin, and respond to follicle-stimulating hormone (FSH). Between the tubules, Leydig (interstitial) cells respond to luteinising hormone (LH) by making testosterone.
The accessory glands differ by species, and examiners ask this constantly. The bull, ram and buck have ampullae, vesicular glands (seminal vesicles), prostate and bulbourethral glands; their ejaculate is small and concentrated. The stallion has all four, and his vesicular glands add a gel fraction. The boar has very large vesicular and bulbourethral glands, which explains his large ejaculate and the gelatinous bulbourethral plugs that are filtered out at collection. The dog has only a prostate (and small ampullae), with no vesicular or bulbourethral glands; the tom has a prostate and bulbourethral glands but no vesicular glands.
Penile type also differs. Ruminants and the boar have a fibroelastic penis with a sigmoid flexure, straightened by the retractor penis muscles' relaxation, so erection adds little diameter. The stallion and the dog have a musculocavernous penis that enlarges greatly. The ram and buck carry a urethral process (filiform appendage) that can obstruct with calculi; the boar has a spiral (corkscrew) glans and a preputial diverticulum; the dog has an os penis and a bulbus glandis that swells to produce the copulatory tie; the tom has androgen-dependent penile spines that fade after castration.
Spermatogenesis in scrotal species needs a testicular temperature a few degrees (about 2–6 °C) below core body temperature. Four mechanisms achieve this: the pampiniform plexus, where cooler venous blood draws heat from the arterial supply by counter-current exchange; the tunica dartos and cremaster muscles, which raise or lower the testes; scrotal sweat glands; and the thin, sparsely haired scrotal skin. Fever, high ambient heat, scrotal dermatitis, fat in the scrotal neck and insulation all cause testicular degeneration.
Spermatogenesis has three phases: spermatocytogenesis (mitotic division of spermatogonia, which renews the stem-cell pool), meiosis (primary spermatocytes to haploid spermatids) and spermiogenesis (the spermatid's transformation into a spermatozoon through the Golgi, cap, acrosomal and maturation phases). Release into the lumen is spermiation. The whole process takes roughly 61 days in the bull, about 49 days in the ram, about 57 days in the stallion and about 39 days in the boar; epididymal transit adds roughly another one to two weeks. The practical consequence is that an insult to the testis shows in the ejaculate for about two months, and a bull must be re-examined about 60 days later before he is condemned.
In the epididymis, sperm gain progressive motility and fertilising ability and the cytoplasmic droplet migrates from the proximal to the distal midpiece and is normally shed; the cauda stores sperm. Hypothalamic gonadotrophin-releasing hormone (GnRH) is released in pulses; each LH pulse is followed by a testosterone pulse. Testosterone (and oestradiol made from it) gives negative feedback on GnRH and LH, while inhibin from Sertoli cells suppresses FSH. Testosterone acts on Sertoli cells, maintains the accessory glands and drives libido.
Erection is parasympathetic, through the pelvic nerves and nitric oxide-mediated filling of the corpus cavernosum; emission and ejaculation are sympathetic, through the hypogastric nerves. After deposition, sperm undergo capacitation (removal of cholesterol and seminal-plasma proteins from the membrane) in the female tract, then hyperactivated motility, and the acrosome reaction on binding the zona pellucida.
A breeding soundness examination (BSE) is a standardised assessment of a male's likely ability to get females pregnant. It does not guarantee fertility and it does not test libido unless a serving-capacity test is added. It has four parts. First, history and a general physical examination: body condition, eyes, teeth, feet and legs (a lame bull will not mount), and signs of systemic disease. Second, examination of the reproductive organs: palpation of both testes for size, symmetry, consistency and pain; the epididymides, especially the tails; the scrotal skin and neck; the penis and prepuce (extended during collection to check for frenulum, warts, injuries and deviation); and, in the bull and stallion, rectal palpation (and ultrasound) of the ampullae, vesicular glands and prostate. Third, scrotal circumference or testicular volume. Fourth, collection and evaluation of semen for motility and morphology.
Testicular size predicts daily sperm production because sperm output depends on the mass of seminiferous tissue. In bulls, scrotal circumference is measured with a flexible tape around the widest part of the scrotum with both testes pulled firmly down. It is moderately to highly heritable and is favourably related to earlier puberty in the bull's daughters, so selecting for it improves the herd as well as the bull. In stallions, the width of both testes or calculated testicular volume is used; in rams, scrotal circumference again; in dogs, testicular width or volume by calliper or ultrasound.
The most widely used bull standard is that of the Society for Theriogenology (SFT, North America). A bull is classified as a satisfactory potential breeder if he passes the physical examination and meets three minimums: scrotal circumference for age (30 cm at 15 months or younger, 31 cm over 15 to 18 months, 32 cm over 18 to 21 months, 33 cm over 21 to 24 months and 34 cm over 24 months), at least 30% progressively motile sperm, and at least 70% morphologically normal sperm. A bull that fails a minimum for a reason that may resolve (youth, recent illness, a poor sample) is classified as 'classification deferred' and retested, usually after about 60 days; one with a permanent defect is 'unsatisfactory'. Other systems exist: Australia's bull breeding soundness evaluation uses its own breed-and-age scrotal tables and reports components separately, and many national schemes differ in thresholds. State which standard you are using.
A serving-capacity or libido test exposes the bull to restrained or oestrous females in a pen for a fixed time and counts mounts and services; it reveals penile deviations and lack of libido that an electroejaculated sample cannot. Example: a bull sold with a satisfactory BSE but which cannot extend his penis is still useless.
For rams, the examination stresses palpation of the epididymal tails for the firm enlargement of epididymitis (Brucella ovis, Actinobacillus seminis, Histophilus somni) and serology for B. ovis where it occurs. For stallions, two ejaculates collected one hour apart estimate daily sperm output, and swabs from the urethra, urethral fossa and penile shaft are cultured for Taylorella equigenitalis, Klebsiella pneumoniae and Pseudomonas aeruginosa as required by national codes of practice. For boars, libido, feet and legs, and semen quality at collection matter most. For dogs, the examination includes prostatic palpation and ultrasound and Brucella canis serology before breeding.
The artificial vagina (AV) is the method of choice for trained bulls, rams, bucks and stallions because it gives a physiological, clean ejaculate. It consists of a rigid outer tube, an inner latex liner, warm water in between, a cone and a graduated collection tube. The liner must be at a temperature slightly above body temperature and lightly lubricated; pressure is adjusted by the volume of water or air. Too cool or too slack and the male may refuse or give an incomplete ejaculate; too hot and the penis is injured. The collection tube is shielded from cold and sunlight, because sudden cooling (cold shock) damages sperm membranes. The male mounts a teaser (a restrained cow, steer, another bull, an oestrous female or a phantom/dummy), the penis is deflected into the AV by the side, and ejaculation in the bull and ram is a single thrust. In the stallion, ejaculation follows several thrusts with rhythmic urethral pulses felt at the base of the penis. False mounts before collection (sexual preparation) increase sperm output in bulls.
Electroejaculation uses a rectal probe delivering rhythmic, gradually increasing electrical stimuli to the pelvic nerves. It is used in bulls, rams and bucks that will not use an AV, in range animals, in wildlife and in injured males. The ejaculate tends to be larger in volume and lower in concentration because of more accessory gland fluid, and morphology is unaffected. It causes discomfort, and some jurisdictions restrict its use or require sedation or anaesthesia for certain species; check local welfare law. Transrectal massage of the ampullae and vesicular glands can produce semen in bulls but is often contaminated with urine and is less reliable.
Other methods include ultrasound-guided transrectal massage with sedation in some species, epididymal sperm recovery after castration or death (valuable for rescuing genetics), and in the tom, urethral catheterisation after alpha-2 agonist sedation (dose — confirm against the product label and your national formulary) or electroejaculation under general anaesthesia.
The boar is collected by the gloved-hand (manual pressure) technique: he mounts a dummy sow, and the collector grasps the corkscrew glans firmly in a gloved hand, applying steady pressure that mimics the sow's cervix. Ejaculation lasts several minutes and comes in fractions: a pre-sperm fraction, the sperm-rich fraction and a post-sperm fraction with gel from the bulbourethral glands. The collection vessel is insulated and covered with gauze or a filter to retain the gel, and the clear first portion is usually discarded because it carries bacteria from the preputial diverticulum. Total volume is typically about 150–300 mL or more.
The stallion is collected with an AV on a mare in oestrus or a phantom; a filter in the AV or a separate filter removes the gel fraction. The penis is washed with warm water only (not soap) before collection, because soaps disturb the normal bacterial flora and favour pathogens.
The dog is collected by digital manipulation, ideally with an oestrous bitch or her scent present. After the bulbus glandis swells, firm pressure behind it and the penis turned backwards allow collection into a funnel and tubes. The ejaculate has three fractions: a small, clear first fraction from the prostate, a white sperm-rich second fraction, and a large third fraction of prostatic fluid. Only enough third fraction is collected for volume; too much prostatic fluid harms chilled and frozen semen.
Rams and bucks are collected with a short AV on a restrained ewe or doe in oestrus; ejaculation is an instant thrust and the volume is about 1 mL with very high concentration. Hygiene at every collection matters: clipped preputial hair, clean equipment, disposable liners where possible and collection in a dust-free area.
Evaluation begins at the collection tube. Volume is read directly. Colour should be milky to creamy in the bull and ram, greyish-white and watery in the stallion and boar. Yellow can be harmless (riboflavin in some bulls) or urine; pink or red means fresh blood (haemospermia); brown means old blood; flecks or clots suggest pus from seminal vesiculitis. Consistency in the bull and ram ranges from watery to thick creamy and rises with concentration.
Mass motility (wave motion) is judged on an undiluted drop at low magnification on a warmed slide, scored on a scale (commonly 0 to 5) for the swirling of dense semen. It is useful only in high-concentration species, the bull, ram and buck. Individual progressive motility, the percentage of sperm moving forward, is judged on a diluted sample under a coverslip at higher magnification, preferably with phase contrast and a slide warmer at about 37 °C. Computer-assisted sperm analysis (CASA) measures velocity and path parameters objectively but must be set up for each species and diluent.
Concentration can be measured with a haemocytometer, a calibrated photometer or spectrophotometer, fluorescence-based cell counters, CASA or flow cytometry. The haemocytometer is the reference method. In the improved Neubauer chamber, the central counting area is 1 mm by 1 mm, divided into 25 medium squares, and the depth is 0.1 mm, so the area holds 0.1 µL. Semen is diluted (traditionally 1:200 in a solution that kills sperm, such as formol saline or water), both sides are loaded and the heads in five medium squares (four corners and centre) are counted. The arithmetic is: count in 5 squares multiplied by 5 (to give 25 squares), by 10 (to give 1 µL), by 1,000 (to give 1 mL) and by the dilution factor. With a 1:200 dilution, the count in five squares multiplied by 10 million gives sperm per mL. Example: 120 heads in five squares at 1:200 gives 120 × 5 × 10 × 1,000 × 200 = 1.2 × 10⁹ per mL. The two sides should agree within about 10%; otherwise the chamber is refilled.
Morphology is assessed on a stained smear (eosin-nigrosin, Williams' or similar) or, better, on a formol-saline-fixed wet mount under phase-contrast or differential interference contrast at oil immersion, counting at least 100 and preferably 200 cells. Two classification systems coexist. Blom's divides abnormalities into major defects (thought to impair fertility more, such as abnormal head shapes, knobbed acrosome, diadem/crater defect, proximal cytoplasmic droplet, midpiece defects such as the Dag defect, and strongly coiled tails) and minor defects (such as distal droplets, detached normal heads and simple bent tails). The older primary/secondary/tertiary system classifies by presumed origin: primary in the testis during spermatogenesis, secondary in the epididymis, tertiary during handling. Reports now commonly list the individual defects, because some, such as the knobbed acrosome and the Dag defect, have known inherited forms in some breeds and some, such as pyriform heads and proximal droplets, rise after testicular insults.
Viability is tested with eosin-nigrosin: eosin enters only cells with damaged membranes, so dead sperm stain pink and live sperm remain white against the dark nigrosin background. The hypo-osmotic swelling (HOS) test shows functional membranes because live sperm take up water and their tails coil. Research and semen-station laboratories add fluorescent probes for membrane integrity, acrosome status (labelled plant lectins), mitochondrial membrane potential and DNA fragmentation (the sperm chromatin structure assay or TUNEL).
Dose calculations follow from total sperm. Total sperm in an ejaculate equals volume multiplied by concentration. Number of doses equals usable sperm divided by the sperm per dose required; the centre decides whether 'usable' means total, progressively motile or progressively motile and normal sperm, so read the basis in each question. Final extended volume equals total sperm divided by the target concentration, and the extender to add equals final volume minus the semen volume. Example: a bull ejaculate of 6 mL at 1.2 × 10⁹ per mL holds 7.2 × 10⁹ sperm; at 20 million sperm per 0.25 mL straw the target concentration is 80 million per mL, so the final volume is 90 mL, the extender to add is 84 mL and the yield is 360 straws. Required sperm per dose differ by species, product and country (conventional frozen bull semen is commonly packaged with roughly 15–20 million sperm per dose, a conventional boar dose with several billion), and national semen standards set minimums; use the standard that applies to you.
An extender (diluent) increases volume so one ejaculate serves many females, and protects sperm during cooling, storage and freezing. A good extender supplies energy (fructose or glucose), buffers pH (citrate, Tris, or zwitterionic buffers), maintains osmotic pressure, protects against cold shock (egg-yolk low-density lipoproteins, milk proteins, or plant lecithin), controls bacteria with antibiotics and, for freezing, contains a cryoprotectant. Classic bovine extenders are egg yolk-citrate, Tris-egg yolk-fructose-citric acid and heated skim milk (heating destroys a milk protein toxic to sperm). Egg-free extenders based on soya lecithin are used where biosecurity or trade rules discourage animal products. Antibiotic combinations such as gentamicin, tylosin, lincomycin and spectinomycin are widely used so that semen meets international health rules; the combination is prescribed by regulation in some markets.
Liquid (fresh or chilled) semen is the main form in pigs worldwide and is used heavily in sheep, goats and horses. Bull semen tolerates cooling to about 5 °C and remains fertile for a few days. Boar sperm membranes are rich in unsaturated fatty acids and poor in cholesterol, so they are very sensitive to cold shock; boar semen is stored at about 15–18 °C in glucose-based extenders, short-term ones for one to three days and long-term ones for about five days or more. Stallion semen is cooled slowly to about 5 °C in skim-milk-based extenders and shipped in insulated containers for insemination within roughly 24–48 hours; individual stallions differ markedly in how well their semen cools, so a trial shipment is standard. Ram semen kept liquid loses fertility quickly and is generally used within about a day of collection. Goat semen contains a bulbourethral enzyme (often called egg-yolk coagulating enzyme, a phospholipase) that reacts with egg yolk or milk to form toxic products; seminal plasma is removed by washing or low egg-yolk or plant-based extenders are used.
Freezing became possible after Polge, Smith and Parkes reported in 1949 that glycerol protects fowl sperm during freezing. Cryoprotectants are permeating (glycerol, ethylene glycol, dimethyl sulfoxide) or non-permeating (sugars, egg yolk, proteins). Cells are damaged during freezing in two ways, summarised as the two-factor hypothesis: if cooling is too fast, water stays inside and forms intracellular ice; if too slow, the cell is exposed too long to concentrated solutes as extracellular ice forms ('solution effects'). The optimal cooling rate balances the two.
For bull semen, a typical sequence is: extension at about 30 °C with part or all of the extender, slow cooling to about 4–5 °C over one to two hours, addition of the glycerol fraction (in two-step methods a glycerol-free fraction A is mixed with an equal volume of fraction B containing twice the final glycerol concentration, so 14% in B gives 7% final), equilibration for several hours at 4–5 °C, filling and sealing of straws, freezing either in liquid-nitrogen vapour a few centimetres above the surface or in a programmable freezer, then plunging into liquid nitrogen at −196 °C. French straws (0.25 mL and 0.5 mL) are the global standard for cattle; pellets frozen on dry ice were used historically. Post-thaw motility of each batch is checked before release; batches below the national standard are discarded.
Storage is in liquid-nitrogen containers ('tanks'). Frozen sperm do not age at −196 °C, but they are damaged each time a straw warms above about −130 °C and refreezes. Rules: keep the canister below the frost line in the neck while searching, take no more than a few seconds, use forceps not fingers, never lift a canister back into the neck after it has warmed, and monitor nitrogen level weekly. Thaw bovine straws in a water bath at about 35–37 °C for at least 30 seconds, dry the straw, cut it and load it into a pre-warmed gun out of the sun and cold. Thawing conditions differ for some products and for equine and canine straws; follow the producer's instructions.
Freezability differs by species. Bull semen freezes well; ram and boar semen freeze poorly and need intrauterine (ram: laparoscopic) or post-cervical deposition and large doses to give acceptable fertility; about a third of stallions produce semen that freezes well, about a third moderately and about a third poorly.
Male infertility is either an inability to mate (impotentia coeundi) or an inability to fertilise (impotentia generandi). The first group includes poor libido (from youth, over-use, pain, obesity, heat stress, social inhibition by dominant males, or low testosterone), musculoskeletal disease (lameness, spinal arthritis and hip disease in older bulls) and lesions of the penis and prepuce. In bulls, the important penile conditions are persistent penile frenulum (a band of tissue tying the glans to the prepuce, often inherited, corrected surgically but such bulls should not be used for breeding stock), spiral (corkscrew) deviation during erection, ventral and S-shaped deviations, penile papilloma (warts) in young bulls, and haematoma of the penis, in which the tunica albuginea ruptures at the dorsal aspect of the distal bend of the sigmoid flexure during a vigorous thrust, producing a swelling cranial to the scrotum. Preputial prolapse is common in Bos indicus and polled breeds with a pendulous sheath. Phimosis (inability to extend the penis) and paraphimosis (inability to retract it) occur in all species; in the dog, paraphimosis after mating or collection is an emergency.
In the stallion, failure to ejaculate, kicking injuries to the penis, and paralysis of the penis (after some sedatives and in debilitation) are seen. In the boar, preputial diverticulum ulceration and penile injuries from other boars occur.
Testicular hypoplasia is a congenital failure of the germinal epithelium to develop; the testes are small and often firm or soft from birth or puberty and do not improve. A recessive inherited form (gonadal hypoplasia) was classically described in Swedish Highland cattle, and chromosome abnormalities such as XXY (Klinefelter-type) males, including tortoiseshell tom cats, are sterile. Testicular degeneration is acquired loss of germinal epithelium from heat, fever, scrotal injury or frostbite, toxins, malnutrition or age; the testes become soft and later small and fibrotic, and sperm show increased head and midpiece defects and proximal droplets. If the cause is removed, recovery takes at least one full spermatogenic cycle plus epididymal transit, so a recheck at about 60 days (bull) is fair.
Orchitis and epididymitis are mostly infectious: Brucella abortus in bulls, Brucella ovis, Actinobacillus seminis and Histophilus somni in rams, Brucella suis in boars, Brucella canis in dogs, and trauma in any species. Brucella infections are zoonotic (B. ovis is an exception in practice) and in many countries notifiable; affected males are culled or, in dogs, managed under specialist and public-health advice. Seminal vesiculitis (vesicular adenitis) in young bulls causes pus and leukocytes in semen and enlarged, lobulated or painful glands on rectal palpation; Trueperella pyogenes and Histophilus somni are among the causes, and response to treatment is often poor. Cryptorchidism is failure of one or both testes to descend; the retained testis does not make sperm but still makes testosterone, so a bilateral cryptorchid is sterile but behaves as a male. It is heritable, common in boars and stallions (the 'rig'), and in the dog retained testes are at higher risk of Sertoli cell tumour, which can cause feminisation and bone-marrow suppression from oestrogen. Segmental aplasia of the Wolffian duct causes obstructive azoospermia on the affected side; sperm granuloma follows rupture of the duct.
Bulls can carry venereal pathogens without signs: Tritrichomonas foetus and Campylobacter fetus subspecies venerealis live in the preputial crypts, which deepen with age, so older bulls are the usual chronic carriers. Diagnosis is by repeated preputial scraping or washing for culture and PCR; control is by testing, culling infected bulls, using young bulls and AI. Bovine herpesvirus 1 causes infectious pustular balanoposthitis. Chromosomal translocations such as the 1/29 Robertsonian translocation reduce fertility in daughters and are screened by karyotyping in AI sires.
In the dog, benign prostatic hyperplasia is the commonest prostatic disease of intact older males and causes haemospermia and tenesmus; castration or anti-androgen drugs (doses — confirm against the product label and your national formulary) reduce the gland. Retrograde ejaculation is confirmed by finding sperm in post-ejaculation urine. Low alkaline phosphatase in seminal plasma, which comes mainly from the epididymis, suggests incomplete ejaculation or bilateral obstruction rather than testicular failure in dogs and stallions.
Artificial insemination was used in the late 18th century by Spallanzani in the dog and developed for farm animals in Russia by Ivanov early in the 20th century; it spread with the artificial vagina, egg-yolk extenders, antibiotics and frozen semen. Its advantages are rapid genetic gain through progeny-tested or genomically evaluated sires, control of venereal disease, no need to keep a bull, use of semen after the sire's death, international movement of genetics and the ability to use sexed semen. Its weaknesses are dependence on accurate heat detection, trained technicians and a reliable liquid-nitrogen chain.
The standard bovine method is the recto-cervical (rectovaginal) technique. The inseminator passes a gloved hand into the rectum, grasps the cervix through the rectal wall, cleans the vulva, introduces the AI gun upward at first to avoid the urethral opening, then guides it through the cervical rings by manipulating the cervix onto the gun rather than forcing the gun. Semen is deposited in the uterine body just beyond the internal os. Deposition deep into one horn, or in the cervix, is less reliable; trauma and contamination reduce conception.
Timing: the cow is in standing oestrus for roughly 12–18 hours on average (shorter in high-yielding dairy cows and longer ranges reported), and ovulates about 10–12 hours after the end of oestrus. Sperm need time to capacitate, and the oocyte is fertile only for a few hours. The practical guide, the morning-evening (a.m.-p.m.) rule from the 1940s, is: a cow first seen in standing heat in the morning is inseminated that afternoon or evening; one first seen in the evening is inseminated the next morning. Activity monitors give time of onset and allow AI roughly 8–16 hours later; once-daily AI is a common farm compromise with modest loss. Buffaloes show weaker signs, more silent heat and seasonal breeding; insemination late in heat, with a second insemination after about 12 hours on some farms, is common practice.
Synchronisation lets a group of females be inseminated together or at a fixed time without heat detection (timed AI, TAI). There are three tools. Prostaglandin F2α (PGF2α) and its analogues regress a functional corpus luteum (CL); they work only from about day 5 to day 17 of the bovine cycle, so either cows with a palpable CL are injected, or all are given two injections about 11–14 days apart so that all have a responsive CL at the second. Progestagens (intravaginal progesterone-releasing devices, ear implants, oral altrenogest in pigs and horses) hold females out of heat while in place and allow a synchronous heat when withdrawn; they also help some anoestrous females to cycle. GnRH (or hCG) releases LH to ovulate or luteinise a dominant follicle and start a new follicular wave. Combined protocols such as Ovsynch (GnRH, PGF2α seven days later, GnRH two days later, then TAI about 16–20 hours later) are standard in dairy herds. In small ruminants, progestagen devices for about 12–14 days plus equine chorionic gonadotrophin (eCG) at removal are widely used, and the male effect (introducing a ram or buck after separation) induces ovulation in the transition season.
The female-reproduction manual covers protocols, their variants and the follicular-wave biology behind them. Drug doses and the legality of products (oestradiol esters, for example, are banned in food animals in some regions) vary by country; every dose — confirm against the product label and your national formulary. In small ruminants, sows and mares, AI timing is covered in the species chapters below.
MOET increases the number of offspring from a genetically superior female. A donor is superovulated with gonadotrophins to make several follicles ovulate, inseminated, and her embryos are collected about seven days later and transferred to recipients or frozen. Two gonadotrophins are used. Pituitary-derived FSH has a short half-life and is traditionally injected twice daily in decreasing doses over about four days; equine chorionic gonadotrophin (eCG) has a long half-life and is given once, but its prolonged action produces more unovulated follicles and higher oestradiol, so its use in cattle has declined. Treatment begins either in mid-cycle (about days 8–12, when a new follicular wave is under way) or, more commonly now, at a planned wave emergence after follicle ablation or oestradiol/progestagen treatment where these are permitted. PGF2α is given on about the third day of FSH treatment to cause luteolysis. The donor is inseminated, typically twice about 12 hours apart, starting about 12 hours after onset of oestrus, often with more than one straw. Doses — confirm against the product label and your national formulary.
Bovine embryos enter the uterus at about day 4–5 after oestrus and are collected non-surgically on day 6–8 (day 0 = oestrus). Under epidural anaesthesia, a two-way or three-way balloon catheter is passed through the cervix into each horn (or the uterine body), the balloon is inflated to seal the horn, and the horn is flushed repeatedly with a buffered medium containing protein; the effluent passes through an embryo filter. Response varies enormously: some donors give none, others dozens; on average a flush yields a modest number of transferable embryos, with the mean and spread depending on breed, age, nutrition and protocol.
Embryos are located under a stereomicroscope and evaluated by the International Embryo Technology Society (IETS) system: a stage code from 1 (unfertilised oocyte) through 2 (2- to 12-cell), 3 (early morula), 4 (morula), 5 (early blastocyst), 6 (blastocyst), 7 (expanded blastocyst), 8 (hatched blastocyst) to 9 (expanding hatched blastocyst); and a quality code from 1 (excellent or good) through 2 (fair) and 3 (poor) to 4 (dead or degenerating). A day-7 bovine embryo should be a compact morula or blastocyst.
Recipients must be healthy, cycling, in good condition and synchronous with the donor, ideally within about one day. Transfer is non-surgical with a sheathed gun into the uterine horn ipsilateral to the CL, under epidural anaesthesia; atraumatic, quick placement in the cranial third of the horn matters. Pregnancy rates are highest for fresh, quality-1 embryos and lower for frozen or poorer ones.
Embryos are frozen by slow freezing in glycerol or in ethylene glycol; ethylene glycol permeates fast enough to allow 'direct transfer', in which the straw is thawed and transferred without removing the cryoprotectant in the laboratory. Vitrification, very fast cooling in high cryoprotectant concentrations so that the solution forms a glass without ice, is preferred for sensitive embryos such as in-vitro-produced and equine embryos.
Embryo transfer is one of the safest ways to move genetics internationally because the intact zona pellucida, correct washing (IETS recommends ten washes, with trypsin treatment for certain viruses) and testing exclude most pathogens. IETS places diseases in categories by the strength of evidence that correctly handled embryos do not transmit them; for bovine in-vivo-derived embryos, category 1 includes, for example, enzootic bovine leukosis, foot-and-mouth disease, bluetongue, Brucella abortus, bovine spongiform encephalopathy and, with trypsin treatment, infectious bovine rhinotracheitis. WOAH's Terrestrial Code sets the trade conditions.
In the mare, superovulation is unreliable, so equine embryo transfer is usually single-embryo: the donor is inseminated, the embryo flushed about 7–8 days after ovulation and transferred to a synchronous recipient. Breed-registry rules decide whether AI and embryo transfer foals can be registered; the Thoroughbred studbooks require natural cover.
Ovum pick-up (OPU) is transvaginal, ultrasound-guided aspiration of oocytes from ovarian follicles. A probe in a vaginal guide carries a needle connected to a vacuum line; the operator holds the ovary per rectum against the probe and punctures each visible follicle, usually those of a few millimetres or larger. OPU can be repeated, commonly once or twice a week, and works in heifers before puberty (from a few months of age), in pregnant cows during the first trimester, and in donors that do not respond to superovulation or have acquired infertility such as blocked oviducts. It can also follow FSH stimulation to increase oocyte yield. Bos indicus cattle typically have more antral follicles and give more oocytes than Bos taurus; buffaloes have fewer follicles and give fewer oocytes.
In-vitro embryo production (IVP) has three steps. In-vitro maturation (IVM) brings the immature oocyte from germinal vesicle stage to metaphase II over about 22–24 hours in a culture medium with gonadotrophins and serum or defined protein; the surrounding cumulus cells expand. In-vitro fertilisation (IVF) co-incubates matured oocytes with sperm, usually frozen-thawed and separated through a density gradient, for about 18–22 hours; heparin is the classic capacitating agent for bovine sperm. In-vitro culture (IVC) of the presumptive zygotes follows for seven or eight days to the blastocyst stage. Typical bovine results are cleavage of most inseminated oocytes and blastocyst development in roughly a third of them, varying widely with laboratory and donor.
In vitro embryos are darker, more lipid-laden and more cryosensitive than in vivo embryos, and serum in culture has been associated with large offspring syndrome (oversized calves, long gestation, dystocia). In recent IETS statistics, in-vitro-produced bovine embryos outnumber in-vivo-derived embryos worldwide, with South America (especially Brazil) and North America the leading users. OPU-IVP uses semen economically, because one straw, including sexed semen, can fertilise oocytes from several donors. In horses, conventional IVF works poorly, so intracytoplasmic sperm injection (ICSI) of a single sperm into each oocyte is the clinical method.
Cloning makes genetically identical animals. Embryo splitting (bisection of a morula or blastocyst) and blastomere separation produce identical twins and a few more; they are limited. Somatic cell nuclear transfer (SCNT) produces copies of an adult. The steps are: a metaphase II oocyte is enucleated; a donor somatic cell, often a cultured fibroblast synchronised in G0/G1 by serum starvation or contact inhibition, is placed under the zona or fused with the enucleated oocyte (cytoplast) by an electric pulse; the reconstructed embryo is artificially activated with chemicals, cultured to the blastocyst stage and transferred to a recipient. Handmade cloning removes the zona and uses manual bisection instead of micromanipulators and has been used widely for buffalo at India's National Dairy Research Institute.
Dolly the sheep, born in 1996 at the Roslin Institute in Scotland and reported in 1997 by Wilmut and colleagues, was the first mammal cloned from an adult somatic (mammary) cell. Cattle, goats, pigs, mice, cats, dogs (2005, Korea), horses (2003, Italy), camels and buffaloes have since been cloned. Efficiency remains low: only a few per cent of reconstructed embryos become healthy live offspring. The causes are incomplete epigenetic reprogramming of the donor nucleus, which shows as early losses, abnormal placentas, hydrallantois, large offspring syndrome and neonatal deaths. A clone is not perfectly identical to its donor: mitochondrial DNA comes mostly from the recipient oocyte, and environment and epigenetics shape phenotype.
Uses include multiplying elite or proven animals (including castrated sport horses and high-merit bulls), conserving endangered breeds and species, and producing transgenic or gene-edited animals for research and medicine. Food from clones and their offspring is regulated differently by country, and welfare concerns about pregnancy losses and dystocia are real; check national law and registry rules before advising a client.
The only method in large-scale commercial use for decades separates X- and Y-bearing sperm by flow cytometry on the basis of DNA content. In cattle, the X chromosome is larger, so X-bearing sperm contain about 3.8% more DNA than Y-bearing sperm. Sperm are stained with a DNA-binding fluorescent dye (Hoechst 33342), passed single-file through a laser beam and oriented so that the flat head faces the detector; the brighter (X) and dimmer (Y) sperm are put into charged droplets and deflected into separate tubes, and dead or unidentifiable sperm are discarded. The method was developed at the United States Department of Agriculture's Beltsville laboratory in the late 1980s. Purity is typically about 90% for the chosen sex. Sorting is slow and damages some sperm, so sex-sorted doses contain far fewer sperm than conventional doses, often around 2 to 4 million per straw depending on the product, and are more expensive. Newer approaches include laser destruction of unwanted sperm in the stream and immunological or other non-flow methods; their claims should be judged on published field data. Sexing of embryos, by biopsy and PCR for Y-chromosome sequences, is an alternative for embryo programmes.
Conception rates with sexed semen are lower than with conventional semen, commonly cited as roughly 70–90% of the conventional rate in well-managed heifers, with larger gaps in lactating dairy cows and under poor management. Newer sorting methods and higher doses have narrowed the gap. Heifers are the best candidates because they are more fertile; good heat detection and insemination at the right time are more critical because fewer sperm are deposited. Some programmes delay AI with sexed semen slightly later in oestrus than with conventional semen, but this varies; follow the producer's guidance and local data.
Uses: dairy herds use X-sorted semen to produce replacement heifers from their best females and conventional or beef semen on the rest (producing more valuable beef-cross calves); beef herds can use Y-sorted semen for steer production; and OPU-IVP programmes use one sorted straw to fertilise oocytes from several donors. In India, sex-sorted semen has been promoted under national schemes to reduce the number of unwanted male calves of dairy breeds, in a setting where cattle slaughter is restricted in many states. The economic case depends on the value of a heifer calf over a bull calf, the extra cost of the straw and the loss in conception rate; the arithmetic must be done for each farm.
Most sheep and goat breeds from temperate latitudes are seasonal short-day breeders: falling day length increases nocturnal melatonin secretion, which releases GnRH pulsatility and starts cyclicity in autumn. Breeds near the equator breed nearly year-round. The ewe's cycle is about 17 days with oestrus of roughly 24–36 hours and ovulation near the end of oestrus; the doe's cycle is about 21 days with a somewhat longer oestrus. Rams and bucks are also seasonal: libido, testicular size and semen quality are best in the breeding season. Out of season, ovulation can be induced by progestagen devices with eCG, by melatonin implants where approved, and by the male effect: rams or bucks introduced after several weeks' separation cause ovulation within a few days, often a silent first ovulation followed by a short cycle, so most ewes show heat about 17–24 days after introduction.
AI is limited in small ruminants by the ewe's cervix, whose misaligned rings prevent passage of a catheter in most ewes. Fresh or chilled semen deposited in the cervical os (cervical AI) gives acceptable results; frozen-thawed ram semen deposited cervically gives poor fertility, so frozen semen is usually deposited directly into the uterine horns by laparoscopy under sedation and local anaesthesia, typically after a progestagen-eCG protocol and at a fixed time after device removal. A notable exception is Norway, where vaginal deposition of frozen semen in local breeds gives acceptable results. Transcervical AI is more feasible in does. Ram BSE stresses the epididymal tails because Brucella ovis epididymitis is common in many countries.
Pig AI uses mainly liquid semen; frozen boar semen gives lower litter sizes and is used chiefly for genetic import and conservation. Gilts reach puberty at about 5–8 months, hastened by daily boar contact. After weaning, sows return to oestrus in about 4–7 days. Oestrus lasts roughly 2–3 days (longer in sows than in gilts) and ovulation occurs about two-thirds of the way through it. Heat is detected by the standing (back-pressure) reflex in the presence of a boar. The common routine is insemination when standing heat is first detected and again at about 12–24-hour intervals while she stands. Conventional (cervical) AI uses a spiral-tipped or foam-tipped catheter locked into the cervix, with a dose of a few billion sperm in about 80–100 mL; post-cervical AI passes an inner catheter beyond the cervix and allows a smaller dose. Boar fertility falls in hot seasons, so boar housing is cooled. Oral altrenogest synchronises gilts; doses — confirm against the product label and your national formulary.
The mare is a seasonal long-day breeder; the stallion is also seasonal. The transitional period in spring brings irregular, prolonged heats with several follicles that fail to ovulate. Artificial lighting, commonly about 16 hours of total light daily begun some 60–90 days before the planned breeding date, advances the season. Oestrus lasts about 5–7 days and ovulation occurs about 24–48 hours before its end, so timing depends on ultrasound: a dominant follicle commonly 35 mm or larger (smaller in ponies, larger in draught breeds), softening, and endometrial oedema. Ovulation is often induced with hCG or a GnRH analogue (doses — confirm against the product label and your national formulary). Fresh or cooled semen (commonly about 500 million progressively motile sperm per dose) is inseminated within roughly 48 hours before ovulation; frozen semen survives a shorter time, so mares are scanned frequently and inseminated close to ovulation, commonly within about 6 hours after it or just before it in an induced protocol. Post-breeding endometritis is normal and transient, but susceptible older mares need uterine lavage and ecbolic treatment.
Twin pregnancies end badly in the mare, so one vesicle is crushed manually when both are found, ideally before fixation of the conceptus at about day 16. Equine viral arteritis is transmitted venereally by carrier stallions, in whose accessory glands the virus persists; contagious equine metritis (Taylorella equigenitalis) is spread by asymptomatic stallions. National codes of practice set swabbing and blood-testing schedules before each season.
The bitch is monoestrous, usually cycling every 5–12 months, with proestrus and oestrus each lasting about 9 days on average but with wide variation. She ovulates primary oocytes that need about two to three days in the oviduct to mature before they can be fertilised. The key event is the LH surge, called day 0; ovulation follows about two days later, and the fertile period is roughly days 4–6 after the surge. Because behaviour and cycle day are unreliable, breeding is timed by serial serum progesterone, which rises from baseline at the LH surge (commonly about 2–3 ng/mL) and reaches roughly 5–8 ng/mL around ovulation; laboratories and assays differ, so use the reference values of the laboratory you use. Vaginal cytology shows an increasing proportion of superficial (cornified) cells through proestrus, near-total cornification in oestrus, and an abrupt return of non-cornified cells and neutrophils at the start of dioestrus (cytological dioestrus), which falls about 8 days after the LH surge (about 6 days after ovulation) and retrospectively dates it; whelping follows about 57 days after the first day of cytological dioestrus. Gestation is about 65 ± 1 days from the LH surge but appears to range from about 57 to 72 days from a single mating because canine sperm can survive for several days in the female tract.
Canine AI uses fresh semen deposited in the cranial vagina, chilled semen deposited vaginally or transcervically, and frozen semen deposited into the uterus, either transcervically with an endoscope or surgically where law permits (surgical AI is prohibited in some countries on welfare grounds). Frozen dog semen survives briefly after thawing, so it is inseminated on the fertile days after ovulation. Before breeding, both dogs are tested for Brucella canis, which causes abortion, epididymitis, discospondylitis and infertility and is zoonotic. Common male problems are benign prostatic hyperplasia, prostatitis, cryptorchidism and testicular tumours.
The queen is seasonally polyoestrous (long-day) and an induced (reflex) ovulator: vaginal stimulation at mating triggers the LH surge, and several matings in a short time are often needed to release enough LH. Oestrus lasts about a week. A non-fertile mating that induces ovulation produces a pseudopregnancy (non-pregnant luteal phase) of about 35–45 days, after which she returns to oestrus; some queens ovulate spontaneously. Gestation is about 63–65 days. Feline AI requires induction of ovulation with hCG or a GnRH analogue and is mostly used in research and conservation of wild felids.
Tom cats are collected with a small AV after training, by electroejaculation under general anaesthesia, or by urethral catheterisation after alpha-2 agonist sedation. Penile spines regress within weeks of castration and their presence in a supposedly neutered cat suggests a retained testis or residual testicular tissue. Tortoiseshell or calico males usually have an XXY karyotype and are sterile.
Contraception and population control are important companion-animal reproductive services: surgical gonadectomy, the deslorelin implant (a GnRH agonist that first stimulates and then suppresses the axis), and progestagens, which carry risks of pyometra, mammary changes and diabetes. Licensed products, doses and age limits vary by country; any dose — confirm against the product label and your national formulary.
Learn the species tables first: accessory glands, ejaculate volume and concentration, site of semen deposition, oestrus length and time of ovulation. Then practise with your hands: collect a bull with an AV, fill a haemocytometer and calculate concentration, stain an eosin-nigrosin smear and count 200 cells, and pass an AI gun through slaughterhouse cervices until it becomes routine. Work at least ten dose calculations on paper (total sperm, doses on a motile basis, extender volume) so that the arithmetic is automatic. Learn the SFT bull classification and be able to classify a bull from a set of findings, because licensing papers present exactly that.
Your daily work is AI, pre-breeding bull and ram examinations and fertility investigations. Run BSEs on every bull before the season and record scrotal circumference, motility and morphology so you can compare across years. Audit your clients' liquid-nitrogen tanks and thawing practice; poor semen handling causes more failures than poor semen. Investigate conception problems in the right order: heat detection and timing, semen handling, technician, female disease, and only then the bull. Advise on when sexed semen pays (heifers, good heat detection) and when it does not. For equine and canine clients, offer ultrasound-timed and progesterone-timed breeding with cooled or frozen semen and know the swabbing and Brucella canis testing rules.
At 25 years you are likely to run or advise a semen station, an ET business or a district breeding programme. Decisions include which sires to collect, how to meet national and WOAH semen and embryo health rules, how to set and audit post-thaw quality standards, and whether to invest in OPU-IVP or sexed-semen programmes. You train and audit AI technicians with conception-rate data by technician and by bull, and you design synchronisation and TAI programmes that fit the farm's labour. You also handle disputes: a buyer's claim that a bull is infertile, or a breeder's complaint about frozen-semen quality, which you resolve with documented, repeatable evaluation.
Research questions remain wide open: why some bulls and stallions freeze poorly, which seminal-plasma and sperm proteins predict field fertility, how to reduce cryodamage and the darker, lipid-rich phenotype of in-vitro embryos, how to raise SCNT efficiency by better epigenetic reprogramming, and how gene editing and genomic selection of embryos will change breeding schemes. A senior scientist designs field trials with enough inseminations per treatment to detect real differences in conception rate, uses objective sperm-function tests rather than subjective motility, and judges new sexing or extender technologies on independent fertility data. Policy work includes conservation cryobanks for indigenous breeds and the welfare and regulatory framework for clones and edited animals.
This manual gives no drug doses: gonadotrophins, prostaglandins, progestagens, GnRH analogues, hCG, sedatives and anti-androgens are named by class only, because labelled doses differ between products and countries. It gives ranges, not single values, for ejaculate characteristics, oestrus length, sperm numbers per dose and progesterone thresholds, because textbooks, laboratories and national standards differ; national minimum standards for frozen semen (such as India's) are referred to but not quoted, and must be checked in the current national document. It does not cover camelids, yaks, mithun, deer, poultry or fish reproduction, gene editing in depth, or the female reproductive cycle and synchronisation protocols in detail (covered in the gynaecology manual) and parturition (covered in the obstetrics manual). Chapter numbers of the WOAH Code are not cited because they change between editions. Field conception rates for newer sexing technologies are summarised only qualitatively.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions.
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