17th Annual "David E. Bartlett"Address
Presented by Dr. Shirley Johnston

Canine Reproduction: Lessons in Reproductive Biology The 2000 Bartlett Address: A Monograph

Prehistoric History of Dogs

Archeological evidence suggests that the dog was the first species to he domesticated by man. With 78 chromosomes, the wolf subspecies are considered the ancestors of the dog in all its breeds, since they have this same number of chromosomes. Dogs today range in weight from 3 Ihs to more than 150 Ibs, making them one of the most diverse species known. A canine mandible dating from about 10,000 BC has been discovered in Western Iran, and it seems likely that some kind of link between the human and the dog was established 12,000 years ago, if not earlier.
This skull, of a Saluki, found in northern Mesopotamia, dates to 3500 BC. The Saluki is a sighthound, known for running down game by sight rather than smell. The sighthound group, which included greyhounds, afghan hounds, Russian wolfhounds and deerhounds was bred for speed, stamina, keen eyesight, and powerful jaws.
Dogs played an important role in Mesopotamian life. This stone relief from the British Museum, dating from about 645 BC, shows the Assyrian king, Assurbanipal, hunting wild onagers on horseback with bow and arrow and dogs.
Greyhounds and wolfhounds also were used in the hunt and as guard dogs in ancient Egypt. The oldest known veterinary publication, the Kahun papyrus of ancient Egypt, dates from 1900 BC, and describes in sacred hieroglyphic script some eye diseases of
dogs, fish and birds. Dogs are seen in paintings, figurines, and mummies dating from ancient Egypt.
This beautiful illustration is from the Joy of the Chase, written in France in the 1300's, more than 100 years before the first book known to have been printed with movable type, the Gutenburg Bible. The author of Joy of the Chase, Gaston Fhehus, devoted 10 chapters of his book to dogs, mainly to the hounds used as companions in the hunt. He suggested that, instead of leaving the care of wounded dogs to prayers directed toward the saints, that owners could apply their own methods of treatment for illness or injury. This book is now in the National Library in Paris.

Discovery of Canine Spermatozoa & Their Role in the Development of Artificial Insemination

Dog sperm played a key role both in the discovery of spermatozoa, and in the discovery of the role of sperm in fertilization. The Dutch habetdasher and microscopist, Anton Van Leeuwenhoek, published hand drawings of canine spermatozoa in Paris in 1679, even though their role in fertilization was unknown until the mid-1800's; in the seventeenth century they were viewed as parasites, or structures that stirred the semen or stirred the sexual desire of the male.

First Artificial Insemination

Lazzaro Spallanzani (1729-1799) a famous eighteenth-century Italian physiologist, held professorships at Modena and Pavia in logic, metaphysics, and Greek. Under the influence of his kinswoman, Laura Bassi, a professor of mathematics, he became interested in science and devoted his leisure time to scientific research. As well as being a scientist and a philosopher, Spallanzani was an ordained priest. Although Arabs may have used artificial insemination (AI) in the horse as early as the fourteenth century, Spallanzani is credited as the first scientist of modern times to achieve pregnancy by artificial insemination, using the dog as a model.
In 1780 he used freshly collected canine semen siphoned from a naturally bred bitch to inseminate another bitch in
heat. Three puppies were born sixty two days later, the first recorded successful AI in any species in modern times.
Initially, Spallanzani supported the prevailing view that spermatozoa were parasites within the semen, and that the germs of all living things were created by God at the time of creation and encapsulated within the first female of each species. Thus, the new individual present in each egg was not formed de novo, but developed as the result of an expansion of parts, laid down by God. It was assumed that semen provided a stimulus for this expansion. However, in semen filtration experiments, Spallanzani showed that fertilization was attributable only to the spermatozoa, or at least, to the solid, filtered elements in the semen.

Long Life Span of Sperm

Dog spermatozoa are notable for other reasons than their use for the first recorded artificial insemination. Unlike the sperm of most mammalian species, which survive in the female reproductive tract and are capable of fertilization tor 24 to 48 hours, clog sperm are very long lived within the reproductive tract of the estrous bitch. This permits fertile copulation many days before ovulation. Motile dog spermatozoa have been recovered from the bitch as late as 268 hours after a single copulation, and motile sperm have been found in the uterine lumen of estrous bitches in undiminished concentrations for 4 to 6 days after a single breeding. Breeding trials suggest that dog sperm have a fertilizing life span of 7-9 days in the reproductive tract of the bitch, even though breedings accomplished many days before ovulation are not optimal, as they result in aged gametes, early embryonic death, and smaller than normal litter size. This tremendous life-span, coupled with resistance to cold shock compared to other species, make dog sperm particularly suited to extension and transport tor chilled semen artificial insemination.
In the 1950's Harrop, Bendorf and Chung shipped dog semen extended with heat treated pasteurized milk from London to New York, California to Hawaii, and London to New Zealand,
witn several conceptions from multiple attempts.

Frozen Semen in the Dog-Surgical Al with Frozen Semen

Use of frozen semen in the dog lagged, substantially, behind this technology in other species, especially development of the bovine AI business in the 1940's and 1950's. First, there are relatively small numbers of sperm in the canine ejaculate, compared to the bovine, and many fewer offspring are desired, mostly by purebred dog fanciers. Second, the canine cervix is an abdominal, not pelvic organ, distant from the vulva, with a narrow and inaccessible cervical lumen, making intrauterine insemination difficult. And third, the value even of purebred puppies rarely justifies the cost in time and money of a frozen semen AI.
Conception with frozen semen in the dog was reported in small studies the 1960's and 1970's, with, in general, poor conception rate and need for large insemination volumes in successful conceptions.
In the early 1980's, Fran Smith, who is here today, began a seties of experiments in our laboratory at the University of Minnesota to investigate semen freezing techniques and performance of frozen semen in the dog. One part of Fran's work involved testing the hypothesis that frozen thawed spermatozoa did not readily traverse the cervix of the bitch when inseminated vaginally.
She laparotomized Beagle bitches, exteriorized the uterus, cervix and cranial vagina, and injected frozen thawed dog semen into the lumen either of the uterine body or the cranial vagina, demonstrating that insemination into the uterus was necessary for normal conception rate and litter size.
This picture shows the first frozen semen litter born to a Beagle bitch at the University of Minnesota. The technique of surgical insemination was used in Fran's work as an experimental tool, and not promoted as the best way to inseminate bitches, but I still remember that it met with great concern worldwide about the ethics of this procedure. Interestingly, by the time I left Minnesota in 1996 the technique was in use both at the Minnesota zoo, where Siberian tigers and other endangered species were being surgically inseminated, either into the uterus or the uterine horn, and also in use in
many clinics arouna tne country inseminating frozen semen. Today, some canine theriogenologists advocate routine surgical insemination of infertile bitches, regardless of whether fresh, chilled extended or frozen semen is used, because anesthesia is easier and safer than it was 20 years ago, and surgical insemination permits examination of the (largely inaccessible) uterus of the infertile bitch. How far we have come.

Discovery of the Canine CL and Its Role in Canine Reproduction

Although investigatots around the world were devoting tremendous energy to the study of frozen semen in dogs in the 1970's and 1980's, discoveries of far greater significance to reproductive success in this species were beginning to be reported at the same time. These began to elucidate the role of the corpus luteum in reproduction in the bitch.
The first of these was the discovery of the hormone assay technique of radioimmunoassay (RIA), for which Rosalyn Yalow received the Nobel Prize in Medicine and Physiology in 1977. This technique grew out of the discovery of antibodies to hormones in the blood of human patients by Yalow and her colleague, Solomon Berson, and the decision to use these antibodies in a competitive protein binding assay to measure minute quantities of substances in biological fluids. This discovery was followed by the establishment of endocrine laboratories, including veterinary laboratories, all over the world. Veterinary colleges, such as those at Colorado State, Cornell, Minnesota and a productive group of Stig Einarson's colleagues at the Faculty of Veterinary Medicine at the Swedish University of Agricultural Sciences began to use RIA to understand animal reproduction.
With funding first obtained by Dr. Ed Mather, and continued by Dr. Brad Seguin, both of whom are here today, the endocrine laboratory at the University of Minnesota CVM was established in 1975, and it went on to provide a tremendous tool in better understanding reproductive biology; and especially, function of the corpus luteum, in research and client-owned bitches. The ability to measure serum progesterone easily and accurately in the bitch led to the discovery that the bitch appears to maintain luteal function for about 2 months after every ovulation, whether or not she is pregnant. Most
otner mammals nave a pregnancy recognition mechanism about 12 to 13 days aftet ovulation that, in the presence of pregnancy, signals continued luteal function and/or pregnancy maintenance; in the absence of pregnancy, the female lyses her corpora lutea, and either initiates another esttus cycle or enters seasonal anestrus. The bitch appears to lack a pregnancy recognition mechanism, always maintaining luteal function for about 2 months whether or not she is bred, or whether or not she is pregnant. Work by Patricia Olson, who is here today, while she was at CSU, and Auke Okkens at Utrecht in the 1980's demonstrated that hysterectomy does not alter duration of luteal function in the bitch, suggesting that pituitary hormones, rather than uterine prostaglandins, appeat to regulate luteal lifespan in this species.
The second major discovery was that canine ovulation may occur at quite variable times in relation to external signs of estrus. In their classic paper defining the onset of diestrus in the Beagle Bitch in 1974, Hoist and Phemister reported on the estrous cycle of 400 Beagle bitches. Even in this single breed, in a relatively uniform colony of research dogs, the day of first estrus was demonstrated to range from 11 days before to 3 days after ovulation, as determined on the basis of the diestrus vaginal smear as an indicator of ovulation day.
This graph depicts the relationship between ovulation day and onset of proestrus in 50 bitches representing multiple breeds managed at our service at the University of Minnesota in the early 1990's. The significance of this broad range of time between onset of estrus or proestrus and ovulation is that optimal insemination time, especially using artificial insemination with chilled or frozen semen, cannot be determined by external reproductive signs.
Finally, came the discovery that preovulatory luteinization occurs in this species and is associated with preovulatory increase in progesterone in peripheral blood that can be used as a predictor of ovulation. Bischoff was the first to describe the folding of the follicle wall of the canine ovary before rupture, and, in 1845, reported that, histologically, luteal tissue in the bitch began to form before ovulation occurred.
Patrick Concannon and his group at Cornell reported in the 1970's that estrogen decline, not peak, was necessary for LH release in the hitch, and that preovulatory secretion of progesterone was necessary for onset of receptive behavior. In Beagles, Pat reported increase above baseline of serum progesterone concentration on the day preceding the LH surge, with discrete serum concentrations on different days around the time of the LH surge. Since bitches are optimally bred 2 to 3 days after ovulation, or 4 to 5 days after the LH surge, detection of increased progesterone any time around the LH surge can be used as a prospective predictor of optimal breeding dates.
This report led, in the mid-1980's to expanded application of serum progesterone testing, using both RIA in laboratories and in house semiquantitative ELISA kits that became tremendously powerful tools in planning breeding management with frozen or extended semen, such as in this Wire Haired Pointing Griffon inseminated with semen imported from France, or in this mother and daughter, both frozen semen puppies from our clinic in Minnesota, or in optimizing conception rate and managing infertility, as in this infertile German Shepherd dog, and this infertile Bichon Frise both treated at our clinic, or in optimizing litter size as in this Skye terrier bitch, as
well as in predicting parturition date in this species.
Even the understanding of the variable response of the canine CL over its lifespan to prostaglandin induced luteolysis, studied by Stefano Romagnoli in Italy, shown here with Patrick Concannon in a recent photo, relies on an understanding of this interesting reproductive tissue, the corpus luteum.
Many observations on canine reproduction, such as the discovery of the first transplantable tumor, the transmissible venereal tumor, by the Russian veterinarian Novinsky in 1876, the physiology and anatomy of the male penis in the interesting canine copulatory lock, the investigation of inherited abnormalities of sexual differentiation in the American Cocker Spaniel in Vicki Meyers-Wallen's work at Cornell, and the occurrence of benign prostatic hypertrophy as a consequence of aging only in dogs and humans despite the fact that all male mammals have androgen dependent prostates, have instructed us in elements of reproductive biology using the dog as a teacher, just as the dog instructed Spallanzani more than 200 years ago.
In the last 25 years we've learned about sperm lifespan, about the canine cervix and its function in frozen semen insemination, and, from radioimmunoassay of steroid hormones and breeding trials, about the canine
corpus luteum, its litespan, and its usefulness in managing canine reproduction.
Current and future studies of reproductive technologies in the bitch, whether these be assisted reproduction or attempts to interrupt reproduction, always will rely on a fundamental understanding of the lessons in canine reproduction advanced in the last 20 to 25 years. I applaud the SFT and ACT for embracing the study of reproductive biology in this species. ~ Thank you.

References

BischoffTLW: Entwicklungsgeschichte des Hunde-Eis. Friedrichh Vieweg, Braunschweig, 1845. as cited by Phemister RD, Hoist PA, Spano JS et al: Time of ovulation in the beagle bitch. Bid. Reprod. 8:74-82.1973.
Cohen, D.: The transmissible venereal tumor of the dog - a naturally occurring autograft? Isr J Med Sci 14:14-19,1978.
Concannon PW, Hansel W, McEntee K: Changes in LH, progesteront and sexual behavior associated with preovulatory luteinization in thi bitch. Biol. Reprod. 17:604-613,1977.
Doak RL, Hall A, Dale HE: Longevity of spermatozoa in the reproductive tract of the bitch. J Reprod Fertil 13:51 -58,1967.
Dunlop, R.H..Williams, D.J.: Veterinary Medicine. An Illustrated History. Mosby, Philadelphia, PA, 1996.
Evans, El: The transport of spermatozoa in the dog. Amer J Physiol 105:287-293,1933.
(70) Hoist PA and Phemister, RD: Onset of diestrus in the Beagle bitch. Definition and significance. Amer J Vet Res 35:401-406. 1974.
Hoist PA and Phemister RD: Temporal sequence of events in the estrous cycle of the bitch. Amer J. Vet Res 36:705-706,1975.
Novinsky MA: Zur frage uber die impfung der kregsigen geschwulste. Zentbl. Med. Wiss. 14:790,1976, Smith, FO: Cryopreservation of canine semen: technique and performance. PhD Thesis, University of Minnesota, 1984.