Repro Lab Practical 2

List the average length of the estrous/menstrual cycle for each of the species below. Cow, mare, ewe, sow, women, dog, cat.

Cow: 21 days, Mare: 21 days, Ewe: 17 days, Sow: 21 days, Women: 28 days, Dog: 6 months, Cat: 17 days

List two characteristics of the dog’s estrous cycle that differ from that of livestock species

Significantly longer in duration, anestrus is a stage, does not have metestrus, estrus stage in dogs is longer than livestock species

Why does the queen’s estrous cycle include a stage known as postestrus?

The cat is an induced ovulator. If the queen does not mate, she does not ovulate and move to the luteal phase, but instead goes into postestrus.

List the type of reproductive cyclicity that each species listed below has. Cow, mare, ewe, sow, dog, cat

Cow: polyestrus, Mare: seasonally polyestrus, Ewe: seasonally polyestrus, Sow: polyestrus, Dog: monoestrus, Cat: polyestrus

Among the livestock species that we discuss, list two characteristics of the mare’s estrous cycle that differ from that of the cow, ewe, and sow (this is not related to seasonality).

Duration of estrus and time from onset of estrus to ovulation in the mare are both considerably longer than for the other livestock species.

List four characteristics of the menstrual cycle that differ from the estrous cycle.

Presence of menses at beginning and end of cycle (no menses in estrous cycle). Ovulation occurs in the middle of the cycle, but at the beginning and end of the estrous cycle. Luteal and follicular phase are equal in the menstrual cycle, but follicular phase is 20% of estrous cycle, and luteal phase is 80%. Fertile period in menstrual cycle is around 6 days, but less than 24 hours in estrous cycle.

What is the primary driver of menopause? List two other species besides women that are confirmed to also undergo menopause.

Depletion of the ovarian pool of primordial follicles. Chimpanzees, orcas, narwhals, and other toothed whale species.

What event needs to occur to initiate the transition from the follicular phase to the luteal

phase?

Luteolysis

List the endocrine events that occur after luteolysis occurs that lead to ovulation.

Progesterone declines, increased release of GnRH from hypothalamus, increased secretion of FSH and LH from anterior pituitary, stimulation of follicle growth, increase secretion of estrogen, threshold level of estrogen is reached and results in positive feedback on surge center of hypothalamus, leads to surge of LH and ovulation.

What occurs after luteolysis that allows for the subsequent endocrine events that lead to ovulation?

Removal of progesterone’s negative feedback on the hypothalamus and anterior pituitary.

List the four methods of semen collection and list species that are typically collected using each method.

Artificial vagina: bull, horse, boar, ram, cat. Electroejaculation: bull, ram, cat, orca. Hand glove/hand cone technique: boar, dog. Manual stimulation: elephant

Describe the components of an artificial vagina and explain how an artificial vagina can work to collect semen.

Components: Rigid, cylindrically shaped casing with n internal rubber lines. Rubber liner of the AV holds water at 42-48 C and can be inflated to provide continuous pressure. The AV mimics the primary sensory stimuli that the glans penis is exposed to upon entry into the female’s vagina (change in pressure and temperature) to stimulate ejaculation.

List the prerequisites for use of an artificial vagina for semen collection.

Male is conscious, male must be trained to mount a dummy (phantom or live dummy animal), male is trained and used to being handled by humans.

Describe the components of an electroejaculation system, including a description of the important features of the probe.

Probe: consists of multiple (2-3) parallel electrodes on the ventral surfaces. The parallel electrodes prevent unwanted contractions. Connection: connects the external end of the probe with electrostimulation unit. Electrostimulation unit: controls the voltage, current, and pulse duration that is delivered to the electrodes.

Explain how the electroejaculation equipment is used to stimulate ejaculation.

The probe is inserted into the male’s rectum with parallel electrodes positioned ventrally against the pelvic urethral region. The electrodes deliver short-low voltage electrical pulses that increase with time. The electric pulses produce a stimulus in the pelvic urethral area leading to stimulation of nerves and subsequent contraction of the muscles of the male reproductive system, as well as the accessory sex glands, ultimately leading to ejaculation.

What technique is most used for semen collection in boars? What is the physiological basis for this collection method?

Hand-glove technique. Pressure made by the hand on the glans penis of the boar mimics the pressure applied by the sow’s cervix during natural copulation. Pressure on the glans penis of the boar is a primary driver of ejaculation.

What are the primary parameters (both gross and microscopic) that are used to evaluate a semen/sperm sample?

Gross evaluation, motility, morphology, concentration

For a breeding soundness exam, what are the two primary sperm evaluation criteria that are assessed? What are the minimum benchmarks for these two evaluation criteria that need to be met for the bull to pass the BSE?

Motility greater than or equal to 30% progressively motile sperm. Morphology greater than or equal to 70% normal sperm.

List types of primary and secondary sperm abnormalities that can be observed.

Primary abnormalities (head): crater defect, tapered heads, ruffled acrosome, knobbed acrosome

Secondary abnormalities (tail): coiled tail, double midpiece, folded tail, detached head

List the components of semen extender and describe their respective functions.

Buffers: prevents marked changes in pH. Lipids: help to maintain the integrity of the sperm cell membrane and help to prevent cold shock. Energy substrates: provide energy for supporting sperm viability and motility. Cryoprotectant: protects cells against damage cause by cryopreservation (ice crystal formation). Antibiotics: help to prevent bacterial growth.

What are the two primary options for semen preservation?

Short-term preservation and long-term preservation.

List the common approaches for semen preservation for cattle, horses, pigs, and humans.

Short-term preservation is commonly used for semen collected from boars and stallions. In this approach, semen is extended, packaged, and then cooled to 5C (stallions) or 18C (boars) and held for a short period- 24-35 hours for stallion semen and 4-6 days for boar semen.

Long-term preservation can be used in several species, but the predominant ones are cattle and humans. For long-term storage, semen is extended, packaged, and gradually cooled prior to being placed into liquid nitrogen (-196C) for indefinite storage.

Describe the possible options for packaging semen for preservation and how this varies among species.

For species where short-term preservation is commonly utilized the volume of semen needed for AI is 20-100 mL so packaging for short-term storage in these species requires larger types of packaging, including syringes, bottles, and bags.

For long-term storage, which requires storage in a liquid nitrogen tank, packaging needs to be more compact and typically involves the use of plastic straws that can hold up to 0.5 mL of extended semen.

What is/are the species?

Cow, mare, sow

What is/are the species?

Cat

What is/are the species?

Human

What is/are the species?

Dog

Identify the components used for bovine semen packaging and storage

A: Goblet, B: Canister, C: Straw, D: Cane

Describe how the components are used for the packaging and storage of bovine semen.

Five 0.5 mL straws are packaged per goblet, two goblets are packaged per cane, canes are held within canisters in an liquid nitrogen tank.

What is the typical volume of semen packaged in a plastic straw for AI of cattle. How about for horses?

Cows: 0.5 mL

Horses: fresh semen—20-40mL, frozen semen—5 mL

List important Do’s and important Don’ts related to semen storage and handling.

Do’s: Keep the tank in a well-lite, well-ventilated area, check the nitrogen level regularly, lock the tank when not in use, securely tie down tank in bed of a truck for transport

Don’t’s: Don’t store tank directly on concrete, don’t store tanks in milkhouse, don’t use a piece of piper to measure nitrogen levels, don’t transport the tank in the passenger seat of a vehicle

Where is the frost line of a semen tank located? What is the significance of the frost line?

The frost line is located approximately halfway up the neck of the liquid nitrogen tank. Above the frost line, temperature rises sharply.

List the supplies needed for proper handling and thawing of a straw of bovine semen for AI.

Forceps, tweezers, scissors or cito cutter, semen thaw bath with thermometer, timer, paper towels, AI gun, AI sheath (appropriate for AI gun being used), gun warmer, semen tank with semen

How long can semen straws be held above the frost line? Why should semen straws not be held above the frost line for longer than this?

10 seconds. Holding semen above the frost line for a long period can lead to transitional warming, which can permanently damage the sperm.

What temperature should bovine semen be thawed at?

35-37C (95-98F)

What is the minimum amount of time that a straw of bovine semen should be held in the water bath for thawing?

30-45 seconds

What steps can be taken during the thawing and straw loading process to help protect sperm from cold shock?

After removing the straw from the water bath, dry it off with a paper towel and hold straw within paper towel in palm of your hand. Pre-warm the AI gun either in a gun warmer or by holding near your body prior to loading the straw. After loading the straw in the AI gun, keep it in a gun warmer or near your body until the AI procedure is performed.

Describe the steps involved in performing the AI procedure in cattle. Begin with the restraint of the cow and end with the site of semen deposition and include all the steps in between.

Restrain the animal in a squeeze chute or head catch. Remove straw of semen from liquid nitrogen tank and place directly into a water bath set at 35-37C for 45-60 seconds. While semen is thawing, make sure the AI gun is being warmed up. After thawing, remove the semen straw from the thaw bath, dry straw thoroughly and then flick to make sure the air bubble is at the end of the straw. Using scissors, cut the heat-sealed end of the straw and then load into a pre-warmed AI gun with the cotton plug end first. Place a plastic protective sheath over the AI gun and lock into place. Make sure to keep the end of the AI gun with semen warm until it is inserted into the female. Clean the perineal/vulvar region of the female thoroughly with a paper towel. Insert the tip of the AI gun into the vulva opening at a 45 degree angle (to prevent entry into urethral opening). Using rectal palpation, manipulate the tip of the AI gun toward the cervical os. Manipulate the cervix to work the AI gun through the cervical rings until the tip is just cranial to the end of the cervix. Once in the correct position, slowly depress the plunger of the AI gun to expel the semen and deposit it in the uterine body.

Explain why the tip of the AI pipette used for AI in pigs is shaped the way that it is.

The tip of the AI pipette has a corkscrew-like shape so that it matches the shape of the sow’s cervix and allows the pipette to be inserted into the cervix during AI.

What is the site of semen deposition when performing AI in the pig? Is it different from the site of semen deposition during natural mating?

Intracervical, same as natural mating

How does the typical volume of semen and amount of sperm used for AI in pigs compare to the cow.

Volume is ~80 mL for pigs as compared to 0.5 mL for cows. The amount of sperm used for AI in pigs is ~2.3 billion while in cows it is ~20 million.

Why is the dose volume and amount of sperm used for AI in pigs so much different than that used for other species?

The sow has a very long and tortuous uterus relative to other livestock species. Large volume of semen stimulates uterine contractions that help with sperm transport.

List some reasons why a livestock producer would choose to utilize artificial insemination.

Maximize genetic potential, reduce mating costs, control reproductive disease, produce offspring from dead or injured sires, improve employee safety.

Compare/contrast the use of artificial insemination in the dairy cattle industry to that of the beef cattle industry.

AI in the dairy industry is heavily utilized- more than 80% of cows are artificially inseminated. AI in the beef industry is significantly less utilized- approximately 10-15% of beef cows are artificially inseminated.

Why do you suppose that AI is more heavily utilized in the dairy cattle industry as compared to the beef cattle industry?

Differences in management- dairy cattle are managed intensively and handled multiple times per day, which makes it easier to utilize AI. Beef cattle are managed more extensively and handled less often, so it is logistically more difficult to implement AI. Differences in cost/benefit ratio for the two industries.

What are the main reasons that use of AI in small ruminant species, such as sheep, is limited?

It is not possible to perform rectal palpation in small ruminants, and their cervixes are difficult to pass which makes transcervical AI less effective. Laparoscopic AI can result in good pregnancy rates, even with frozen-thawed semen, but the procedure requires special skills and equipment and it is expensive.

List one similarity and one difference between performing AI in the mare as compared to the cow.

The AI procedure in both species is transcervical. Rectal palpation is used for cattle, whereas the AI pipette is guided through the cervix in the mare by placement of a hand intravaginally.

List the site of semen deposition during AI versus natural mating in the cow.

Natural: vagina, AI: uterine body

List the site of semen deposition during AI versus natural mating in the mare.

Natural: Cervix/uterine body, AI: uterine body

List the site of semen deposition during AI versus natural mating in the sow.

Natural: cervix/uterine body, AI: cervix/uterine body

List the site of semen deposition during AI versus natural mating in the ewe.

Natural: vagina, AI: cervix, uterine body, uterine horn (method dependent)

List the site of semen deposition during AI versus natural mating in the dog.

Natural: vagina, AI: vagina, uterine body

List the site of semen deposition during AI versus natural mating in the cat.

Natural: vagina, AI: vagina, uterine body or horns

What is this?

Artificial vagina

What is this?

Electroejaculation equipment

What is this?

Cito cutter

What is this?

Thaw unit (water bath)

What is this?

AI gun

What is this?

Paper towels

What is this?

Thermometer

What are these?

Semen straws

What is this?

AI gun warmer

What is this?

Tweezers