The double helix structure of DNA begins to breakdown in some people and animals as they age. The degree of fragmentation
of sperm DNA correlates very highly in humans, with the inability of sperm to initiate a birth regardless of the technology
used to fertilize the egg such as artificial insemination (AI), in vitro fertilization (IVF) or intra-cytoplasmic sperm injection
(ICSI). Additionally, sperm with fragmented DNA can fertilize an egg, but embryo development stops at an early stage, and
early pregnancy loss commonly occurs. Statistics from some older stallions confirm this as mares will become pregnant, but
consistent embryo loss will occur at or around the same time of gestation. This concept of DNA fragmentation now is clearing
up many cases of previously unexplained infertility. DNA fragmentation has little to do with the para- meters measured in
a standard semen evaluation and is a totally independent variable. Varner suggests that a sperm chromatin assay be performed
on all older stallions and on any stallion with a fertility question. This test currently is being done at a number of veterinary
schools and private labs. A semen sample is collected and shipped on dry ice to the lab. The sample is thawed and treated
by stress exposure to a low-pH solution. The sample then is labeled with an orange dye that binds exclusively to the broken
or fragmented DNA. A flow cytometer then views the sample and uses a special wavelength of light to measure the amount of
orange (fragmented DNA) verses green (intact DNA), and a computer plot yields a DNA fragmentation index (DFI). Stallions that
show normal breeding behavior and acceptable semen analysis but that have a poor DFI are likely "over the reproductive hill."
Very little in the way of technological help can be given to these stallions, and their breeding careers are likely over.
Varner advises veterinarians to discuss with clients the possibility of freezing sperm from these and other older stallions.
"Technology is advancing rapidly and it is very likely that in the not-too-distant future science will be able to utilize
methods to repair DNA and to use frozen sperm itself in methods of high-tech regeneration and pregnancy production," Varner
Currently, many breed associations do not allow such technology, but should science advance and current rules be changed,
then many older stallions would be able to contribute their DNA to far future generations.
Whit Byers, a partner in Select Breeders Southwest, a company that uses mobile reproductive laboratories to provide breeding
and reproductive services to the equine industry, is more concerned with what can be done for some older stallions in the
here and now. Byers says there are very few stallions that can't have their productivity improved and very few that are truly
too old to reproduce. He agrees with Varner that a sperm chromatin assay is an extremely important part of the work-up of
an older stallion, but if that assay shows a low DFI, then he recommends centrifugation of semen samples from stallions with
low motility or low sperm counts. Centrifugation at 300 G to 350 G for 10 minutes to 12 minutes and then re-suspension of
the pellet in extender produces a highly concentrated low volume of semen. Byers next recommends using a more focused breeding
delivery approach for insemination. Some veterinarians use a flexible infusion pipette and attempt to inseminate as close
as possible to the end of the horn near the breedable follicle. Byers takes this "deep-horn insemination" procedure one better
and believes that using a video endoscope further improves the breeding odds. He uses a 3/10-cc insemination dose of concentrated
semen deposited at the uterine-tubal junction.
"The use of highly concentrated semen applied at precisely the right place" has produced good results in some stallions that
were previously problem breeders and whose reproductive careers were almost over, Byers says.
Breeds that do not allow artificial insemination or technical manipulation cannot use this procedure because of its invasive
nature. Therefore, fewer older Thoroughbred stallions continue to breed compared to older Quarterhorse, Arab and other breed
stallions. But more new tests continue to be developed that will help identify specific sperm-related problems and solutions
surely will follow close behind what will benefit all breeds. Genetic variability always will ensure that some stallions,
for any number of reasons, are born infertile or lose their reproductive ability as they age, and some stallions just keep
on going strong. Veterinarians often are asked to evaluate these older stallions, and good sperm analysis and knowledge of
these new tests and procedures will allow more complete and accurate recommendations.
Dr. Marcella, a 1983 graduate of Cornell University's veterinary college, was a professor of comparative medicine at the University
of Virginia. His interests include muscle problems in sport horses, rehabilitation and other performance issues.