The Informed Breeder:
Semen Collection and Packaging for Chilled Semen Shipment
By Valeria Rickard, DVM
The American Kennel Club allows breeders to perform their own collections for chilled semen shipments. However, AKC does not provide training or continuing education classes for breeders on how to do this properly, and unfortunately we have had many samples arrive at our veterinary hospital where the quality of the sample was very poor, sometimes due to imperfect collection methods and sometimes simply because the packaging was very poor. We have had semen arrive in lunch boxes; sometimes the sperm were all frozen and dead because it was in direct contact with an ice pack; and other times there was no semen present at all in the sample that was sent.
I am hoping this article will be of use to explain the different nuances involved in semen collection and proper sample preparation for sending a chilled semen sample.
A quality chilled semen shipment is dependent on two primary things: 1) you must have collected a quality sample before you package, and 2) you must take care in packaging it correctly so that it is well protected and will arrive in good condition for the breeding. For breeders who do a lot of collections and shipments, or even a moderate number, it makes sense to invest in some equipment. One piece of equipment I highly recommend is a microscope. Microscopes are available in a wide range of prices, from less than $100 to several thousand, but it isn’t necessary to spend a fortune to have one that will allow you to evaluate sperm on a slide. Most of the rest of the supplies you’ll use are disposable, and I want to emphasize “disposable.” I know sometimes breeders try to clean and disinfect supplies, but water and detergents can kill sperm, so anything that is not perfectly rinsed and disinfected can be detrimental to future collections. You will increase your rate of success by using and then replacing supplies that are designed to be disposable.
COLLECTING THE SAMPLE
These elements are essential to a good collection:
- Location – If at all possible the semen collection should be performed in an environment familiar to the male, and this familiar environment should be quiet and free of distractions during the collection. Make sure that the area has a good, non-slip flooring to minimize any chance for slips, sprains or injuries.
- Male Preparation – “maiden” males, those that have never been used before, may be very nervous and reluctant when a semen collection is first attempted, and sometimes training sessions may be beneficial prior to the required collection date. Walk the male about 30 minutes prior to collection and allow him to urinate adequately. It is best NOT to allow male to urinate IMMEDIATELY prior to collection to minimize urine contamination. Lastly, before the collection begins make sure to clean the male’s sheath area well so there is little or no discharge present on the prepuce (the loose fold of skin that covers the glans of the penis). The sheath may be cleaned using water and a paper towel or gauze.
- Motivation – a “teaser” bitch (female in estrus) should be used to optimize the quality of the sample. If that is not possible, satisfactory collections can be recovered using stored frozen swabs, gauze or pads containing the scent from an estrus female.
The Collection Process
Bring the estrus female into the room first and allow her to get comfortable. Then allow the male to enter. Have both the dog and bitch on a leash, but do not use training collars or chokes; you don’t want either animal to feel that they are being corrected as if they are doing something wrong.
Allow the male to sniff the hindquarters of the female so to arouse his libido. Depending on the male’s experience, he may try to mount the female and start performing thrusting motions. If he does not initiate this on his own, a manual stimulation of the penis needs to be applied. As erection begins, the skin of the prepuce needs to be pushed back over the bulb of the penis (the round-shaped swelling towards the end of the penis). A sterile collection bag (Whirl-Pak bag), collection cones or an artificial vagina may be used to collect the semen sample.
The person collecting the semen must encircle the penis and the bulb tightly with thumb and forefingers, simulating a tie. The male will continue to thrust and will give a sample. The male may attempt to step over the collector’s arm as he might during a real tie, and should be allowed to do so.
Three Fractions to the Collection
There are three fractions, or parts, to the collection. The first one is a clear fluid (a pre-sperm fraction which is usually discarded). The second one is the thicker, milkier sperm-rich fraction – this is the one that we need to collect – and it is followed by a clear third fraction which is mostly prostatic fluid, which is not necessary for the sample preparation. Once the male starts to give this last, clear prostatic portion of the collection, release the grip over the bulb and gently remove your collection bag.
Post Collection Care of the Male
Sterile KY jelly can be gently applied to the penis to minimize the potential for cuts or trauma and to speed up detumescence. Prior to kenneling, make sure that the male’s penis is fully back within the prepuce. Make sure the skin is not rolled inside-out and that no hair is stuck around the penis.
EVALUATING THE SEMEN
It is very important to include a semen evaluation form with a chilled semen shipment. If you’d like to attempt to evaluate your stud dog’s sperm at home, you will place one drop of semen onto a pre-warmed microscope slide and examine under the microscope for an overall motility (movement) and morphology estimate. These are the characteristics of the semen that need to be evaluated:
- Color – Normal semen should be a “milky” color. If the ejaculate is red or brown, this indicates the presence of blood. If it is a yellow color, it is from urine. If it is green, it is from pus.
- Volume (ml of ejaculate) – This measurement can be extremely variable, depending on the male’s age, breed and libido. Variations are from one ml to greater than 20 mls.
- pH – Measured on the prostatic (third) fraction only, pH can be measured with litmus paper. Normal should be around 6.5 to 7.0, and abnormalities may indicate infection or inflammation.
- Progressive motility – The measurement of overall percentage of the sperm that are alive and moving. Normal motility should be greater than 70 percent.
- Morphology (structure) of sperm cells – It is difficult to see structural differences in sperm without a good microscope, special staining of the sperm, and some training. Special phase-contrast microscopes are very helpful, but expensive, and are primarily used in reproductive labs. Eosin-nigrosin or DiffQuik stains can be used in the home environment, but again they require training. “Normal morphology” for sperm is when more than 80 percent of them appear structurally within normal range. (See sidebar.)
- Sperm Count – Very beneficial to know, but may be difficult to achieve in the home environment. Automated sperm counters can be purchased, or a hemocytometer can be used to perform a count. A hemocytometer, originally developed for counting blood cells, is a special type of slide with an indention in its center that creates a chamber where cells, including sperm cells, can be suspended in liquid. A grid is etched into the glass of the hemocytometer, and the number of cells in a square can be counted and used to calculate overall sperm count. By one of these methods it can be determined whether the sperm count is low, average or abundant.
HOW DOES HIS SPERM LOOK?
An important part of determining a stud dog’s soundness as a producer is an evaluation of sperm morphology – the size and shape of sperm – when his semen is viewed under a microscope. Normal canine sperm have an oval-shaped head and a long tail. Sperm that are abnormal may have a misshapen, large, small or detached head, or a crooked, split, detached or curled tail, or may have a droplet attached to the tail, and these abnormalities will likely affect the ability of the sperm to reach and penetrate the egg.
What causes sperm to become abnormal? Many factors may influence a dog’s fertility at a specific point in his life, and a qualified veterinarian can be very helpful to a breeder who experiences issues with a stud dog’s fertility. However, the breeder himself can become more familiar with what normal and abnormal sperm samples look like by examining them under a microscope.
Sperm cells may have abnormalities associated with either “spermatogenesis,” or the creation of the sperm cells in the testes, or abnormalities associated with the transport of the sperm from the testes all the way to the egg. When abnormalities are associated with the creation of the sperm, they may be congenital (existing at birth, but not hereditary) or acquired. Acquired conditions may include infections such as brucellosis or a urinary tract infection, inflammation of the prostate due to infection or disease, testicular cancer, prolonged disuse of an intact male, or possibly damage to the testicles that may be related to medications or injury. A dog that is being campaigned under a heavy show schedule may be stressed enough that his sperm morphology will be abnormal for a period of time, and on occasion a dog that is used excessively may have sperm abnormalities for a time.
Dogs with abnormal sperm evaluations frequently recover, so repeating an abnormal sperm evaluation is recommended, depending on the suspected cause of the issues. It is important to determine whether the morphological problems are associated with the transport of the sperm or the health of the dog itself, as transport issues can often be addressed by modifying your procedure.
Below are some examples of morphologically abnormal cells.
PACKAGING SEMEN FOR SHIPMENT
- After collecting the semen from the male you will put the sperm-rich “second” fraction only into a pre-warmed Whirl-Pak bag and measure the total volume collected using a syringe. It is preferred to use syringes – warmed up to room temperature – that have no rubber in them. The volume can alternately be measured by placing it into a warmed 15 ml sterile plastic tube.
- Carefully follow the manufacturer’s directions for the extenders being used. Semen extender is a liquid diluent added to the semen to preserve its fertilizing ability. On average, semen is extended at a 1:3 to 1:4 dilution ratio (one part semen to three parts extender or one part semen to four parts extender) with an extender that has been warmed to approximately 37° C. Draw the needed volume of extender into a syringe and slowly add the extender to the semen, drop by drop, over a period of three to five minutes.
- Close the lid of the tube tightly, and, you can also use Parafilm or tape to secure the lid in place. Label the tube with the dog’s name, owner’s name, breed and today’s date. It is VERY important that you label the tube as well as the bag that it is placed in! Place the sealed tube with semen into a small Whirl-Pak bag and close tightly. This extra bag will help keep the sperm safe if something happens to the tube during the transport. (The semen could still be recovered for breeding if it leaked into the bag.)
- Place the sealed bag into a small Styrofoam container. Use either a rubber band or scotch tape to secure it in place, and wrap the container in paper towels.
- Prepare your shipping box based on manufacturer’s recommendations. Remember that the shock of both heat and cold kills sperm! Place ice packs in appropriate places, and make sure that the ice packs are well insulated by newspapers so they are not in direct contact with the sperm container. Make sure to include the evaluation report, then seal container well and attach a FedEx or UPS label. (If semen is being shipped for a weekend breeding, make sure to mark it for Saturday delivery.) Write down the tracking number so you can look it up on the Internet in the event that there are issues with the semen being delivered on time. Good luck!
This article originally appeared in Terrier Type 2011 Stud Register and is posted here with permission.