Artificial insemination (A.I.) as practiced by bees and many other flying insects has played an important role in plant reproduction for a very long time.
Use of A.I. in animals is a human invention and more recent. Undocumented tales exist of Arabs obtaining sperm from mated mares belonging to rival groups and using the sperm to inseminate their own mares. However, our story starts with recorded history, where facts are available to document noteworthy achievements. Consequently, the story is related chronologically.
The development of A.I. is a remarkable story of tireless workers dedicated to the pursuit of knowledge, to the replacement of fiction with facts and the application thereof. This review can only provide a taste of the important discoveries and developments associated with A.I. and the people most involved. A more comprehensive overview of the technical aspects are available in many of the books on A.I. and reproduction.
Early history of A.I.
Leeuwenhoek and his assistant, Hamm, were the first persons to see sperm which they called “animalcules.” Leeuwenhoek did not have an advanced formal education, so he did not study Latin, the scientific language of the day. However, he was a clever, capable individual who ground lenses so precisely that sperm were visible. His published paper amazed, and perhaps amused, the reigning king of England, who regularly read papers submitted to the Royal Society, where Leeuwenhoek’s paper was published.
Another century passed before the first successful insemination was performed by Spallanzani in a dog which whelped three pups 62 days later. Spallanzani originally trained to be a priest, but he had a great interest in natural history and pursued the latter. He was a professor of natural history in Pavia by the age of 25. He collected, analyzed and classified a large array of butterflies, shells and other marine and land animals.
Another 100 years passed before Heape and others in several countries reported that A.I. had been used in isolated studies with rabbits, dogs and horses. Heape was an outstanding reproductive biologist, establishing much of the basis for the relationship between seasonality and reproduction. This led to Cambridge becoming a world center for reproductive studies.
A.I. becomes a focal point of research
Pioneering efforts to establish A.I. as a practical procedure were begun in Russia in 1899 by Ivanow. By 1907 Ivanow had studied A.I. in domestic farm animals, dogs, foxes, rabbits and poultry. Some of this research, especially in horses, is included in a paper in English submitted June 21, 1922 and published in record time in the July 1922 issue of the Journal of Agricultural Science. He developed semen extenders and trained technicians to select superior stallions and multiply their progeny through A.I.
Much of the A.I. work in Russia was taken over later by Milovanov, described in a text translated into English. He established major projects for sheep and cattle breeding. In his own workshop Milovanov designed and made practical artificial vaginas and other items, many similar to those used today. This was an enormous improvement over the earlier method of collecting semen from sponges placed in the vagina of mount animals.
The development of A.I. by Ivanow also stimulated research outside of Russia. The Japanese scientist Dr. Ishikawa studied with Ivanow. He returned to Japan and began a similar program in horses in 1912. This gradually developed into A.I. being applied in Japan in cattle, sheep, goats, swine and poultry. Other Japanese researchers became involved. Because most of the research was published in Japanese and few Westerners knew Japanese, little was known about this research in the Western world until Niwa and Nishikawa summarized the research in English.
News of the extensive use of A.I. in Russia following the Ivanoff report became widespread in the Western world with the publication of the book on A.I. by Walton. Walton conducted a number of experiments, including a pioneering shipment of ram semen to Poland, which 2 days later was used for successful insemination of ewes. However, commercial A.I. did not evolve rapidly in the United Kingdom.
Some A.I. work, particularly with horses, had been performed in the early 1900s in Denmark. Eduard Sorensen, at The Royal Veterinary College in Copenhagen, Denmark, was familiar with the Russian work. With Gylling-Holm, Sorensen organized the first cooperative dairy A.I. organization in Denmark in 1936. The program enrolled 1,070 cows the 1st year and 59 percent conceived, slightly better than natural service in the same herds. This was an important stimulus for the development of A.I. in dairy cattle in the United States and other Western countries.
The Danish veterinarians established the method of rectovaginal fixation of the cervix, allowing semen to be deposited deeply into the cervix or into the body of the uterus. This technique provided a tremendous advantage because fewer sperm were required for insemination of each cow. Another Danish “invention” was the straw for packaging semen. In 1956, I saw some of the original oat straws that Dr. Sorensen kept in his desk. Subsequently he saw children at a birthday party for his daughter sipping punch with cellophane straws, and he recognized that he had found the straw that he needed. Later Cassou produced straws commercially that have been used worldwide.
Meanwhile, the much earlier research by Spallanzani led eventually in Italy to the development of an artificial vagina for dogs by Amantea in 1914. This work served as a model for the Russian development of artificial vaginas for bulls, stallions and rams. Another Italian, Bonadonna, continued research on A.I. in several species. His enthusiasm for the potential value of A.I., along with Lagerlof, resulted in the establishment of the highly successful International Congress on A.I. and Animal Reproduction held every 4 years. The first one was held in Milan in 1948.
In Sweden, Lagerlof became known for his research on infertility problems in bulls. This research was stimulated by his visit with W. W. Williams, a Cornell DVM, who had published methods of staining spermatozoa. Meanwhile, Lagerof completed his classic PhD dissertation titled “Changes in the spermatozoa and in the testes of bulls with impaired or abolished fertility”. He established a group with worldwide influence in training veterinarians in the various aspects of fertility and A.I.. Other Scandanavians, such as Blom, followed, publishing a steady stream of excellent papers on abnormal sperm morphology. These pioneers were all thinkers and doers, and they trained many who followed.
Modern development of A.I. in dairy cattle
Phenomenal growth of A.I. occurred in the 1940s in the United States. The procedures developed in the United States became established worldwide. In 1936, Brownell was inseminating cows in the Cornell herd, and other A.I. work was started in the late 1930s in Minnesota and Wisconsin. In 1938, an A.I. cooperative was established in New Jersey, modeled after the Danish system. Another one in 1938 followed in the state of New York.
The development of the New York Artificial Breeders, Cooperative, Inc., currently Genex, Inc., in Ithaca, New York made possible the close collaboration between a farmer cooperative and researchers and extension personnel at Cornell University. This was a highly productive relationship resulting in the experimental insemination of hundreds of thousands of cows and publication of more than 100 research papers on sire selection, testicular evaluation, semen collection, evaluation and processing, and fertility testing.
The most widely used test of sperm quality from the initial stages of A.I. development until the present time has been the assessment of the proportion of normal, progressively moving sperm. Thus, a good microscope is the key.
With frozen semen, evaluation of post-thaw survival became important. Ejaculate volume and sperm concentration are the two other critical components of semen evaluation because they determine the number of sperm obtained. Volume originally was measured in graduated containers. Volume today often is determined more accurately by weight.
Fertility of sperm is the ultimate test of sperm quality. Often it is not possible to measure fertility, so many tests of semen quality in addition to motility and morphology have been correlated with fertility. For commercial A.I., an inexpensive method of estimating fertility, based on cows not returning for insemination, was developed as an essential component of the A.I. program. This made possible the comparing of fertility of bulls, inseminators, semen processing procedures and even herd performance under practical field conditions. It provided a remarkable new system of recording breeding efficiency.
Semen extenders, semen cooling and extension rate
Initially the most important problem to resolve was a method to store semen long enough for shipment and use in the field. The first major improvement in the A.I. procedure initiated in the United States was the development of a yolk-phosphate semen extender. Salisbury et al. improved the media by buffering the egg yolk with sodium citrate. Sperm survival at 5°C permitted use of the semen for up to 3 days, and the citrate dispersed the fat globules in egg yolk, making sperm visible for microscopic examination. This semen extender was used worldwide for cattle. Glycerol was added later for cryopreservation of bull sperm.
The next major stimulus to A.I. of dairy cattle was an improvement of about 15 percent in fertility resulting from a better method of initially protecting sperm from cold shock and the control of some venereal diseases by the addition of antibiotics. The Cornell extender, containing the antibiotic mixture of penicillin, streptomycin and poly myxim B, was used for many years as the standard.
Many years were required to eradicate the diseases from bulls. During that time in vitro treatment of semen with antibiotics prevented transmission of several diseases. Antibiotics are still included as “insurance” protection against possible contamination.
With A.I. expanding rapidly, demands for semen from popular bulls increased. The simplest way to meet this demand was to “stretch” each ejaculate farther by using fewer sperm per insemination, providing that this could be accomplished without sacrificing fertility. Salisbury and coworkers published several classic papers clearly supporting the concept that only a few million sperm per insemination were required. In conducting these experiments Salisbury was criticized by some who declared that “dilution” of semen was like “watering the milk.” Consequently, Foote and Bratton introduced the word “extender” because the yolk-citrate-antibiotic medium enhanced and extended the usefulness of semen. This word has “stuck.”
The net result of these experiments was that semen extension could be increased at least 25-fold. Sperm numbers per insemination with liquid semen were reduced from more than 100 × 106 sperm per insemination to 4 × 106 sperm per insemination.
The yolk-based extender was improved with Cornell University Extender (CUE), which resulted in the highest fertility achieved in A.I. on hundreds of thousands of inseminations. Shannon visited Cornell in the late 1950s and modified the extender for use with liquid semen in the intensive breeding season in New Zealand. Caproic acid and catalase were included with 5 percent egg yolk by volume to form “Caprogen,” an effective extender for preserving bull sperm at the moderate ambient temperatures of New Zealand, with as few as 2 × 106 sperm per insemination.
Several researchers had reported that the volume of egg yolk used originally could be greatly reduced, particularly at ambient temperatures, and that catalase might be beneficial at room temperature. Milk also was widely used. Following the report by Michajilov, Almquist and coworkers published a series of papers on skim milk and whole milk, establishing the optimal procedures for detoxification of milk and addition of glycerol for freezing semen. The milk-glycerol extender continues to be used by many A.I. organizations.
Bull sexual behavior, intensity of semen collection and sire power
To meet the high demand for semen from selected bulls, harvesting the maximal number of sperm per bull was the other approach to increasing the number of inseminations possible per bull. Early research on sexual preparation and intensity of semen collection was initiated by Bratton and colleagues and Almquist and colleagues. The extensive studies by Almquist and associates included beef bulls as well as dairy bulls.
These collective studies resulted in recognizing the importance of evaluating the sexual responses of individual bulls and applying stimuli along with an optimal frequency of about six semen collections per week. The result was a sperm output of 30 to 40 × 109 sperm per week per sire. With a 50-week-per-year collection schedule and 10 × 106 sperm per insemination dose, these sperm numbers translate into 200,000 doses of semen for insemination each year.
Large differences occurred in sperm output of bulls when semen was collected under comparable conditions. Methods were developed to evaluate both the quality and quantity of spermatogenesis in bulls. These studies revealed that the differences in sperm output were largely due to testicular size. Testis size was easily measured and was highly inherited. So, attention to testis size was important in selection and evaluation of sires.
Extensive studies were conducted on nutrition, performance and aging of bulls. Rapid growth was important in minimizing the time required for young bulls to reach puberty and be tested for A.I. Unfortunately, these data are largely unknown because they were published in bulletin form, although one classic paper appeared.
Genetic selection of bulls for milk
One of the major reasons for initiating A.I. was to make the males that transmit superior genetics for milk production available to more producers in the animal industry. This was democracy in action. The elite bulls would not be limited to the wealthy. However, bull selection procedures practiced before the A.I. era gave disappointing results. Many bulls with high proofs in natural service did not repeat in A.I.
Robertson and Rendel in Scotland and Henderson at Cornell University pioneered new methods of sire selection. Henderson continued his research to establish the principles required for optimal sire selection programs and to provide objective methods for adjusting records for unequal environmental influences.
While the geneticists were making breakthroughs on sire selection, an astounding achievement was reported from England, the successful freezing of chicken sperm by including glycerol. Glycerol soon was found to be useful for bull sperm. Many researchers had tried to freeze bull semen but had failed. A bit of serendipity played a role in the discovery.
The research had focused on using sugars as cryoprotectants, but they did not lead to successful results. However, Polge relates that he returned six months later to try again, and results were promising, presumably with the same bottle of fructose stock solution. Why success now? What was in the bottle? Chemical analysis showed that the bottle contained no sugar, but rather glycerol and protein in proportions comprising Meyers albumin used for histology. Apparently, there had been a mistake in labeling when reagents were stored.
The basic medium used by Polge was the original yolk-citrate extender plus glycerol. Almquist and coworkers developed whole milk-glycerol as a good medium to cryopreserve bull sperm. Tris-buffered egg yolk-glycerol also provided excellent protection for sperm either frozen or unfrozen. This soon became the most commonly used medium worldwide for cryopreservation of bull sperm and sperm from several other species.
Packaging frozen semen for use with solid carbon dioxide (dry ice ) or liquid nitrogen was a problem. Glass ampules often broke during freezing or thawing. Cassou modified the system developed by Sorensen with a method for sealing plastic straws and a gun for insemination. Originally 0.5-mL capacity straws were used, but 0.25-mL straws are popular because they require less storage space.
Another major change in storage occurred in the 1950s with the shift from solid carbon dioxide storage at −79°C to liquid nitrogen at −196°C. Researchers had demonstrated that sperm survival at −196°C was virtually infinite, whereas biologic changes occurred with storage at −79°C. Also, storage with solid carbon dioxide was not convenient, and frequent resupply was necessary.
Liquid nitrogen storage also was a problem, because insulation of the tanks was inefficient. Frequent refilling was required to maintain a safe temperature of about −196°C. Manufacturers of tanks were not interested in improving tanks until J. Rockefeller Prentice, owner of American Breeders Service, privately provided a substantial sum of money, which convinced Linde Division of the American Cyanamid Company that there was a market for liquid nitrogen containers with improved insulation. The successful cryopreservation of sperm and development of efficient liquid nitrogen containers provided the foundation upon which today’s entire cryopreservation industry is built. PD
References omitted but are available upon request at firstname.lastname@example.org
—Excerpts from Cornell University Extension website