ARTIFICIAL INSEMINATION ON FARM ANIMALS

Term Paper for Ag. Dev.: Rural Development, Strategies and Approaches

KILATON, JUN IAN C.

February 24, 2021

Introduction

The livestock sector is one of the fastest growing segments of the agricultural

economy particularly in the developing world (Delgado et al., 2009). Over the past decades,

the Philippine livestock and poultry industry have been consistently contributing positively

to the economy even with lesser support from the government compared to other

agricultural commodities. Thornton (2010) describes livestock sector in developing

countries like the Philippines as evolving to respond to rapidly increasing demand for the

livestock products. The rapidly growing population of the country and increasing

purchasing power of the consumers continue to drive the economic potential for these

industries.

Animal breeding has been practice across the country for the improvement of

desirable genetic traits or enhance the traits of the animal in the next generation. Different

methods of animal breeding has been applied but the application of Artificial Insemination

(AI) has been considered as a promising tool to improve genetic potential of farm animals,

yet, many farmers field conditions are unaware about the technology with huge regional

variations in terms of knowledge level and adoption of this promising technology (Foote,

2002).
 AI plays an important role to increase the yielding capacity of cows and is the

appropriate and cheapest way of genetic improvement and the realization of breeding

programs has to be well organized and excited in a very reliable way and AI is fully

functional when it is corporated with good animal husbandry such as effective heat

detection (Noakes, 2009). AI has proven to be a very effective reproductive technology that

selectively increases genetic gain through increased selection pressure on males. Farm

animals, males as well as females, are usually chosen for breeding programs based on

breeding soundness examinations (BSEs). These BSEs determine suitability and likelihood

of females or males to participate successfully in breeding programs. Estrus cycles of

females can be manipulated to institute efficient insemination programs. With the use of

these estrus synchronization programs, large groups of females can be inseminated at the

same time. This does not only have the advantage of concentrating work on specific days

during breeding (Webb, 2003). Another reason for AI is to ensure effective use of semen.

An increased number of offspring from a superior sire can be produced when AI is

employed. Freezing bull semen can provide up to 200 straws of frozen semen from one

ejaculate, equaling 200 AI doses. Overuse of males is prevented and commercial

distribution facilitated. Other important aspects are the prevention of venereal disease

transmission, for example, Trichomonosis and Campylobacteriosis both of which decrease

reproductive efficiency through decreased pregnancy rates, high return rates to estrus and

increased pregnancy losses, that plays a major role in the economic system of offspring

production, and increased safety for valuable breeding animals as mating related injuries

are avoided. Furthermore, AI can be used for frozen semen from males that have died or

are not physically available for mating due to distance or physical inability (Gamborg,
2005). In livestock rearing, the producer makes efficient use of the generous supply of

sperm available from an individual male in a manner that greatly increases genetic

progress, as well as improving reproductive efficiency in many situations. Today, many

bulls have been reported to produce sufficient semen to provide enough sperm for 40,000

breeding units in one year (Bearden et al., 2004).

OBJECTIVE

1. This paper aims to review the recent study carried out on the artificial

insemination techniques and management applied for farm animals.

 FUTURE TRENDS IN ARTIFICIAL INSEMINATION

It is highly probable that the use of AI in livestock will continue to increase. AI not

only facilitates more effective and efficient livestock production, but can also be coupled to

other developing biotechnologies, such as cryopreservation, selection of robust

spermatozoa by single layer centrifugation, and sperm sex selection.

AI in increasing the efficiency of livestock production. Apart from some specialist

sheep or goat units focusing on milk production for cheese and intensive meat production,

farming of these species tends to be confined to marginal land that is unsuitable for crop

production or grazing for dairy cattle. There has been limited selection for production

traits. However, there is a resurgence of interest in them now in developed countries

because of growing awareness that small ruminants could represent better utilization of

scare resources than larger ones, such as cattle, while producing less methane and effluent.

In many developing countries, sheep and goats are better suited to the climate than cattle,

and it is culturally acceptable to eat their meat and milk products. Thus it is likely that

there will be an upsurge in the use of AI in sheep and goats in the future, with an emphasis

on improving production traits by the introduction of superior genes. However, it is

essential that any A.I. scheme aimed at large scale improvement of the national herd must

be supported by improved animal husbandry and animal health, otherwise the pregnancies

resulting from AI will not go to term, and the offspring will either not survive or will fail to

thrive. Many of the advanced ART are of little help in areas where basic husbandry skills

are inadequate.
Biomimetic sperm selection

One potential disadvantage of AI is that the natural selection mechanisms within the

female reproductive tract to select the best spermatozoa for fertilization may be bypassed

when AI is utilized. Biomimetics is the use of technologies and/or processes that mimic a

naturally occurring event. Several in vitro procedures have been suggested that could be

used to mimic selection of good quality spermatozoa in the female reproductive tract and

thus fit the definition of biomimetics in ART. These include sperm processing procedures

such as swim-up, sperm migration, filtration and colloid centrifugation (reviewed by

Morrell & Rodriguez-Martinez, 2009). Of these methods, the one that is most applicable to

livestock and human spermatozoa is colloid centrifugation.

Density gradient centrifugation

Spermatozoa for fertility treatment are usually processed to remove the seminal

plasma and to select those of better quality. In most cases, this is achieved either by sperm

migration, in which the more motile spermatozoa are separated from the rest of the

ejaculate, or by density gradient centrifugation, where the most robust spermatozoa are

selected. The benefits of density gradient centrifugation are as follows (Morrell, 2006):

o Poorly motile and abnormal spermatozoa are removed,

o Sources of ROS (cell debris, leukocytes, epithelial cells and dead or dying

spermatozoa) are removed;

o Sperm survival is improved during frozen and non-frozen storage;

o Bacterial contamination is controlled without antibiotics.

Single layer centrifugation

Density gradient centrifugation is seldom used when processing animal semen

because of the limited volume of semen that can be processed at one time and the time

taken to prepare the different layers. A novel sperm preparation technique, Single Layer

Centrifugation (SLC) through a colloid, was developed at the Swedish University of

Agricultural Sciences (SLU) to select the most robust spermatozoa from ejaculates. This

method is similar to density gradient centrifugation (DGC), but is better suited for animal

semen since it has been scaled-up to process whole ejaculates. The major applications for

SLC-selection are similar to DGC end have been reviewed extensively by Morrell &

Rodriguez-Martinez (2010)

Sex selection

For many centuries, animal breeders and researchers have endeavoured to control

the sex of the offspring born, for various reasons. Initially male offspring were preferred for

meat production, because of the better feed conversion efficiency and lean-to-fat ratio of

males, whereas females were preferred for dairy purposes, except that some males of high

genetic merit were still required as sires. Couples may want a child of a specific sex to avoid

the expression of sex-linked disorders.

Many methods have been proposed for separating X- and Y-chromosome bearing

spermatozoa, based on physical properties, e.g. size of the sperm head, or functional

properties e.g. swimming speed. However, the only method which has been shown to work

reliably is that of selection and separation of spermatozoa whose DNA is stained with a bis-

benzimidazole dye, H33342, using the sorting capacity of a flow cytometer (Morrell et al.,
1988; Johnson et al., 1989). This method functions because the X chromosome is larger

than the Y, therefore taking up more of the DNA-specific stain and showing a higher

fluorescence when the spermatozoa are passed through a laser beam. In bulls, for example,

the difference in DNA content between the X and Y- chromosome is approximately 4.2%.

However, the process of sorting sufficient numbers for an insemination dose in the flow

cytometer takes too long, since the stained spermatozoa must pass one at a time through a

laser beam for detection of their DNA content. Moreover, the pregnancy rate after

insemination of sexed bull spermatozoa is lower than with unsexed spermatozoa, making

the procedure inefficient and expensive. Experience has shown that the staining profiles

are highly individual, with the result that it is not possible to separate the X- and Y-

chromosome bearing spermatozoa efficiently from all males.

Alternative methods of sex selection are also being investigated. A company in

Wales, Ovasort, has identified sex-specific proteins on the sperm surface and have raised

antibodies to them. It is intended to use the antibodies to aggregate spermatozoa bearing a

specific sex chromosome, thus enabling them to be removed from the general population.

A combination of ARTs would also be relevant for sperm sexing. Thus, the speed of

flow sorting can be increased by first removing the dead and dying spermatozoa from the

population, for example by density gradient centrifugation or single layer centrifugation.

Such a combination may increase the “sortability” of sperm samples. Sufficient sexed

spermatozoa may be obtained from flow sorting for IVF, thus generating embryos or

blastocysts for subsequent transfer. However, methods of speeding up the selection

process are needed if flow cytometry is to become useful for species other than the bovine.
Sperm cryopreservation

As previously mentioned, the ability of cryopreserved spermatozoa to retain their

fertilizing ability varies widely between species. New cryoextenders and new protocols are

being developed constantly in an effort to address this issue. One recent advance has been

the introduction of dimethylsulphoxide and the amides formamide and dimethylformamide

as cryoprotectants, in place of glycerol. These molecules seem to function better than

glycerol for some individuals whose spermatozoa do not freeze well, for example, some

stallions. One explanation for this observation is that these molecules are smaller than

glycerol and therefore may cause less damage when they penetrate the sperm membrane.

However, no method appears to be universally successful within one species. As far as

turkey spermatozoa are concerned, it seems that the development of a successful freezing

method will require more than new cryoprotectants and additives (Holt, 2000).

Removal of viruses from ejaculates

Viral infectivity can be removed from the semen of patients with viral infections

such as HIV and hepatitis, by a sequential method of sperm preparation i.e. centrifugation

on a density gradient followed by a “swim-up” (reviewed by Englert et al., 2004).

Spermatozoa from virally infected men prepared by this method have been used in assisted

reproduction attempts, apparently without sero-conversion of mothers or children.

However, some studies with HIV report that density gradient centrifugation alone will not

remove all viral infectivity (Politch et al., 2004). Since spermatozoa may function as vectors

for viruses (Chan et al., 1994), further work is required to investigate how closely different

viral particles are associated with the sperm membrane with putative carry-over during
processing. The double method of processing has also been successful in removing equine

arteritis virus from an infected stallion ejaculate in a preliminary study (Morrell &

Geraghty, 2006). SLC together with swim-up was used to reduce viral infectivity from boar

semen spiked with porcine circo virus 2 (Blomqvist et al., 2011).

AI in conservation biology

It has been suggested that AI and other forms of ART could be useful for genetic

conservation and preservation of rare breeds. Many of these technologies have been

successful to some degree in a research setting, but none have produced results sufficient

to effect population-wide improvements in genetic management (Morrow et al., 2009).

Cryopreservation of semen has been the most widely applied ART in this respect, but much

of the frozen semen in so-called gene banks has never been tested for fertility. A lack of

suitable females or dearth of knowledge about the reproductive biology of the species

involved may contribute to this deficit. However, long-term storage of frozen gametes of

unknown fertility is not a sustainable policy for the conservation of rare breeds and

endangered species. The development of in vitro methods of testing sperm fertility would

contribute considerably to conservation efforts. Since the semen quality in these animals

may be poor (Gamboa et al., 2009), techniques such as SLC of samples prior to AI could be

of considerable benefit in conservation breeding.

 DEFINITION AND HISTORY OF ARTIFICIAL INSEMINATION

Artificial insemination (AI) or introduction of semen in the female genital tract by

means of instruments is the first generation of reproductive biotechnologies which was

feasible in cattle. It is a process by which sperm are collected from the male, processed,

stored and artificially introduced into the female reproductive tract for the purpose of

conception (Webb, 2003; Temesgen et al., 2017). The first commercial AI cooperative was

established in 1936 by a Dane, Sorenson (Foote, 2002). Before the Second World War, most

cows in Europe and North America were fertilized by means of natural service. However,

since several cows on different farms were mated by the same bull, the spread of genital

diseases with decreased fertility outcomes was a constant threat. Moreover, keeping herd

bulls was expensive and represented potential danger for the herd manager (Vishwanath,

2003). Apart from these facts, the limited number of offspring produced per bull after

natural mating made it impossible to set up effective progeny testing schemes and resulted

in a very poor genetic gain. The introduction of AI in cattle was mainly forced by sanitary

reasons, and especially by fertility problems caused by Campylobacter foetus subspecies

venerealis (vibriosis) and Trichomonas foetus. However, also the control and prevention of

non-sexually transmitted diseases such as tuberculosis, brucellosis and paratuberculosis at

the farms benefited from the introduction of AI (Thibier and Guerin, 2000). Semen is

collected from the bull, deep-frozen and stored in a container with Liquid Nitrogen at a

temperature of minus 196 degrees Centigrade and made for use. Artificial insemination has

become one of the most important techniques ever devised for the genetic improvement of

farm animals. It has been widely used for breeding dairy cattle as the most valuable

management practice available to the cattle producer and has made bulls of high genetic
merit available to all (Webb, 2003; Bearden et al., 2004; Temesgen et al., 2017). In livestock

rearing, the producer makes efficient use of the generous supply of sperm available from an

individual male in a manner that greatly increases genetic progress, as well as improving

reproductive efficiency in many situations. Today, many bulls have been reported to

produce sufficient semen to provide enough sperm for 40,000 breeding units in one year

(Bearden et al., 2004). The first successful insemination was performed by the Italian

physiologist and priest Abbe Lazzaro Spallanzani (1780) in a dog which whelped three

pups 62 days later (Foote, 2002). And over 100 years later, in 1890, it was used for horse

breeding. According to (Webb, 2003), the history of AI is interesting in that old Arabian

documents dated around 1322 A.D. indicate that an Arab chieftain wanted to mate his prize

mare to an outstanding stallion owned by an enemy. He introduced a wand of cotton into

the mare‟s reproductive tract, and then used it to sexually excite the stallion causing him to

ejaculate. The semen was introduced into the mare resulting in conception. In 1899, Ivanoff

of Russia pioneered AI research in birds, horses, cattle and sheep, and was apparently the

first to successfully inseminate cattle artificially. Mass breeding of cows via AI was first

accomplished in Russia where 19,800 cows were bred in 1931 (Webb, 2003; Temesgen et

al., 2017)
 PRINCIPLES OF ARTIFICIAL INSEMINATION

In Britain, AI in dairy cattle began to be available in 1942, and by 1950 20% of dairy

cattle were being inseminated. By 1960, more than 2 million cows were inseminated

yearly, which was about 80% of the maximum level that AI would reach (Brassley, 2007).

The established procedure for AI in cattle since the 1960s is transcervical deposition of

semen into the uterine body. This technique replaced the original vaginal or shallow

cervical insemination performed in the 1940s as the intrauterine method proved to be

more efficient and resulted in higher fertility (Lopez-Gatius, 2000). Evaluation of female

gynaecological All females of reproductive age in a herd must be submitted to

gynecological examination for selection of suitable animals for artificial insemination

program. This is an internal examination by rectal palpation, ultrasound and vaginoscopy

and can be complemented by laparoscopy and biopsy. On rectal palpation and

ultrasonography are checked the size, consistency and contraction of the uterus, uterine

horns and symmetry. In the ovaries are observed consistent form and size of follicles, cysts

and persistent corpusluteum. Vaginoscopy complements rectal palpation and

ultrasonography, because it turns out the shape of the vaginal portion of cervix, the

opening degree of the cervical canal, mucosa color, moisture content and characteristic

vaginal and cervical mucus. Gynecological examination involves a complete evaluation of

all components of the external and internal genitalia, with emphasis on the ovaries,

combining the findings of the examination with a score of animal body, with its history and

with the herd (Antonio et al., 2011). Bull health control Disease prevention in bulls has

been considered as essential as in breeding females and new bulls need to be screened by a

qualified veterinarian for infectious agents prior to entering a new herd. Bulls have been
recommended to be purchased only from reputable seed stock producers with adequate

herd health plans; including vaccination against infectious diseases, e.g. leptospirosis and

campylobacteriosis. Bulls are also recommended to be tested annually for brucellosis, but

not be vaccinated for brucellosis. In some instances, bulls need to be vaccinated for bovine

viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and trichomoniasis (Hansen,

2006). The frequency of tests made and the diseases tested at NAIC are not sufficient

(Agegnehu, 2007). According to the international animal health code of the Office

International des Epizooties (OIE)), donor and teaser animals should be tested for the

following specific diseases: Bovine Brucellosis, Bovine Tuberculosis, Bovine Viral Diarrhea,

Infectious Bovine Rhinotracheitis, Campylobacter fetus/subspecies veneralis, Trichomonas

fetus (OIE, 2001). Investigation of bull fertility Infertility or sterility has been accepted as

common problem in the male as in the female but because of the greater hazards presented

by parturition and pregnancy, acquired infertility or sterility, however, is much more

frequent in the female. Bulls selected for AI have been shown to transmit to their offspring

the genetic potential for well-above-average milk or meat production (Herman et al., 1994).

In addition, the progeny must be of desirable conformation, be long wearing, have quiet

disposition, and be free of genetic defects (Herman et al., 1994). Evaluating sexual desire

(libido) which can be affected by age, heredity, environment and poor feeding retards its

onset (Hansen, 2006). Full libido may be achieved before normal spermatogenesis and

therefore, as a rule animals are not put to stud until a few months after puberty. Bulls

retain normal sexual desire until five or six years of age, but beyond this point libido very

gradually wanes. Inability to perform service despite normal sexual desire is a frequent

cause of bull infertility, has been reported to be due to skeletal or visceral pain, in others to
lesions of the genital organs, inability to protrude and penile deviations, while in many

cases in which no lesions can be found the nervous control of copulation is believed to be

defective (Arthur et al., 1983).

Other considerations Scrotal circumference provides a good indication of a bull's

ability to produce sperm and is related to his own age at puberty. The measurement should

be taken at the largest diameter of the scrotum. Both testicles should be positioned next to

each other and a flexible measuring tape should be placed snugly around the scrotum.

Testicles need to be descended into the scrotum, and should be of the same size and shape.

Any irregular shape or swelling may indicate abnormal structure, illness, or injury (Hansen,

2006).
 IMPORTANCE OF ARTIFICIAL INSEMINATION

Artificial insemination Artificial insemination is an essential technique in breeding

programs with progeny testing. AI provides the opportunity to choose sires that proven to

transmit desirable traits to the next generation and minimizes the risk of spreading

sexually transmitted diseases and genetic defects. So far, AI using frozen semen has played

an important role in increasing genetic progress by upgrading the reproductive rate of the

male. It increases the selection intensity since less bull is needed and this is the basis for

selection progress (Tadesse, 2010). AI plays an important role in enhancing animal

productivity, especially milk yields, in developing countries that have a well-defined

breeding strategy and a sound technical base to absorb and adapt the technology to meet

their needs (BBC, 2015). Daughters of AI sires produce significantly more milk than those

of herd bulls sires and the income from this extra milk may cover the extra costs resulting

from extended calving intervals because of low heat detection. A study indicated that

daughters of AI sires were producing almost 900 kg of extra milk per lactation than

daughters of natural service bulls (Valergakis et al, 2007). Another report from USA

showed a difference of more than 1000 kg of milk per lactation on farms using AI (Smith et

al., 2005; Temesgen et al., 2017). This means that farming using AI can be more profitable

apart from covering the extra costs even with calving interval of 13.5-14 months

(Valergakis et al, 2007). Another of the major advantages of artificial insemination is the

elimination of the costs and dangers of maintaining a bull on the farm. The use of AI is the

cumulative beneficial effects on dairy cows because of the opportunity of choosing sires

that are proven to transmit superior genetic traits. The risk of spreading sexually

transmitted diseases or genetic defects is also decreased when AI is practiced on a dairy

farm. Natural mating allows transmission of venerealy transmitting diseases like

brucellosis, listerosis, leptospirosis, trichomoniasis etc between males and females (IAEA,

2005). Some pathogens can be transmitted in semen through artificial insemination, but

the collection process allows for the screening of disease agents. The progeny testing can

be done at an early age. The semen of a desired size can be used even after the death of that

particular sire. The semen collected can be taken to the urban areas or rural areas for

insemination. It makes possible the mating of animals with great differences in size without

injury to either of the animal. It is helpful to inseminate the animals that are refusing to

stands or accept the male at the time of estrus. It helps in maintaining the accurate

breeding and calving records and increases the conception rate. It helps in better record

keeping. Old, heavy and injured sires can be used (Johnson, 2011). Collected semen is also

routinely checked for quality, which can help avoid problems associated with male

infertility (Chatikobo, 2009).

PROBLEMS ENCOUNTERED ON ARTIFICIAL INSEMINATION

Despite the well-known advantages of artificial insemination, a large number of

dairy farmers all over the world still use natural service (NS) bulls to breed their cows. The

main arguments allegedly justifying their choice are higher AI costs compared to those of

keeping herd bulls and additional costs resulting from extended calving intervals because

of low heat detection rates when AI is used. AI costs include; labor, equipment, liquid

nitrogen, semen and three ratios of “services per conception” (Valergakis et al, 2007). The

availability of economically priced liquid nitrogen for the cryopreservation of semen is also

a particular constraint to utilize AI as a whole (FAO International Technical Conference,

2010). Artificial insemination requires accurate time of insemination to ensure the best

chances of conception. The whole reproductive success of a stud farm can be reliant on the

skills of inseminators and there is room for human error. Artificial insemination is a trained

skill, taking a lot of time and practice to carry out efficiently and effectively each time.

Because of this, a qualified vet or animal technician will be needed and these can be costly

(Thomas, 2011). Other disadvantages of AI include poor conception rates due to poor heat

detection, low efficiency of AI technicians and dissemination of reproductive diseases

(GebreMedhin, 2005). High cost of collection, processing, storage and transport of semen,

as well as budget and administrative problems and inefficiency of AI technician is also

another disadvantage of AI (Desalegn, 2008). When receiving semen from other state or

country, the timing becomes even more imperative, as it needs to arrive within the correct

time frame to thaw out and place in the cow. AI can be quite labor intensive when it comes

to lining up the cow to inseminate and so becomes costly due to regular vet checks. AI

decreases the value of stock and increases chances of cattle being larger in bred (Shehu et

al., 2010). Artificial insemination can be limiting if the proper resources are not available,

so there are some disadvantages. AI requires specialized knowledge, trained individuals,

and the time required to properly execute an effective AI program is considerably more

than with natural service. The extra help and time can often mean added expense (Gentry,

2010).
 EARLY HISTORY OF ARTIFICIAL INSEMINATION

In 1899 the first attempts to develop practical methods for artificial insemination

were described by Ilya Ivanovich Ivanoff (Russia, 1870-1932). Although Ivanoff studied

artificial insemination in domestic farm animals, dogs, rabbits and poultry, he was the first

to develop methods as we know today in human medicine. He was a pioneer in the

selection of superior stallions multiplying their progeny through AI. The work of Ivanoff

was taken over by Milovanov, another Russian scientist. He published his paper on

“Artificial insemination in Russia” in the Journal of Heredity in 1938. Milovanov established

major projects for cattle breeding and designed the first artificial vaginas, very similar to

those used today.

The innovating work in Russia inspired Eduard Sörensen from Denmark to organize

the first cooperative dairy AI organization in Denmark in 1933, followed by the

introduction of the first AI cooperative in the US in 1938 by EJ Perry, a dairyman from New

Jersey. In the US and other Western countries the number of AI cooperatives increased

rapidly. Nowadays more than 90 % of dairy cows are artificially inseminated in the

Netherlands, Denmark and the United Kingdom. November 1, 1939, the first animal, a

rabbit, conceived by artificial insemination was exhibited in the United States at the 12th

Annual Graduate Fortnight at the New York Academy of Medicine. Gregory Pincus, an

American biologist, removed an egg from the ovary of a female rabbit and fertilized it with

a salt solution. The egg was then transferred to the uterus of a second rabbit, which

functioned as an incubator. Dr. Pincus conducted his experiments at Harvard University.

 Considering humans, only after the introduction and availability of donor sperm,

artificial insemination became very popular (AID). For many years homologous artificial

inseminations were only indicated in cases of physiologic and psychological dysfunction,

such as retrograde ejaculation, vaginismus, hypospadias and impotence.

With the routine use of post-coital tests other indications were added such as hostile

cervical mucus and immunologic causes with the presence of antispermatozoal antibodies

in the cervical mucus.

The first reports on human artificial insemination originated from Guttmacher

(1943), Stoughton (1948) and Kohlberg (1953a; 1953b). It was the real start of a new era

in assisted reproduction.

Other important research discoveries in animal studies undoubtly influenced the

development of artificial insemination, also in human. Phillips and Lardy (1939) were the

first to use egg yolk to protect bull sperm cells from temperature shock upon cooling. This

protection was explained by the effect of phospholipids and lipoproteins in the egg yolk.

Salisbury et al. (1941) improved the media by using egg yolk with sodium citrate,

permitting the use of semen at 5° C for up to three days. Polge and co-workers (1949) were

the first to freeze fowl and bull spermatozoa by using glycerol in the extender media. In

1950 Cornell University scientists (New York) discovered the benefit of antibiotics added

to the sperm solution in artificial insemination processes. The so-called Cornell extender

(Foote and Bratton, 1950) contained the antibiotic mixture of penicillin, streptomycin and

polymyxim B and was used for many years as the standard. Antibiotics are still used for the

protection against possible contamination.

 In 1953 Dr. Jerome K. Sherman, an American pioneer in sperm freezing, introduced

a simple method of preserving human sperm using glycerol. He combined this with a slow

cooling of sperm, and storage with solid carbon dioxide as a refrigerant. Sherman also

demonstrated for the first time that frozen sperm, when thawed, were able to fertilize an

egg and induce its normal development.

As a result of this research, the first successful human pregnancy with frozen

spermatozoa was reported in 1953. Considering the hostile climate for DI at the time (the

Cook County Supreme Court ruled that artificial insemination with donor semen was

contrary to public policy and good morals) it is not surprising that nearly a decade passed

before the first successful birth from frozen sperm was announced in public, a major

breakthrough in history.

Considering all these new developments, it could be expected that in the 1970s the

sperm bank industry became very popular and commercialized, especially in the United

States.
 DEVELOPMENT AND USE OF ARTIFICIAL INSEMINATION

Artificial insemination for Beef Cattle

Beef cattle greatly outnumber dairy cattle in the United States. The technology of

semen handling and insemination of beef cows is similar to that used for dairy cows, but

beef cows are not managed as conveniently for AI. Many cows are on extensive ranges

where detection of estrus and rounding up animals in estrus for insemination is not cost-

effective. Therefore, the proportion of beef cattle bred by AI is low (Foote, 1981; Dziuk and

Bellows, 1983). Where small groups of beef cows are kept in close confinement, estrous

cycleregulating agents can be used to synchronize the time of insemination for at least one

insemination. In crossbreeding programs, AI has the advantage because semen can

inexpensively replace maintaining bulls of separate breeds.

Artificial insemination for Swine

The AI of swine was initiated by Ivanow in Russia in the early 1900s (Ivanow, 1907;

Ivanoff, 1922). More extensive investigations were conducted in the 1930s (Milovanow,

1938; Anderson, 1945; Maule, 1962; Nishikawa, 1964). Early work was started in the

United States in Missouri (McKenzie, 1931), in Japan in 1948 (Ito et al., 1948; Niwa, 1958),

and in western Europe in the 1950s (Polge, 1956). Boars are easily trained to mount

dummies (Milovanov, 1938; Polge, 1956). All artificial vaginas developed for boar semen

collection provided a means of applying pressure to the glans penis (McKenzie, 1931; Ito et

al., 1948; Polge, 1956), or a gloved hand can be used directly. McKenzie trained many early

outstanding reproductive physiologists (Andrews, Casida, Phillips, etc.). He attended

animal science meetings long after officially retiring, asking questions about the latest
developments. At the 75th meeting of the American Society of Animal Science, when a roll

of long-time members was called, he was the only member standing at the end, a member

for 61 yr. Russian diluters for boar semen were based on glucose solutions with sodium

potassium tartrate or sodium sulfate and peptone, keeping the concentration of

electrolytes low (Milovanov, 1938; Anderson, 1945; Polge, 1956; Maule, 1962). The

recommended storage temperature was 7 to 12°C. However, Ito et al. (1948) recommended

storage at 15 to 20°C. When the yolkphosphate, yolk-citrate, and milk extenders were

developed for bull semen, they were used or modified for boar semen (Polge, 1956; Niwa,

1958; Maule, 1962), including use with cooled semen. Major efforts were made to freeze

semen, following the successful use of frozen bull sperm (Nishikawa, 1964; Graham, 1978;

Iritani, 1980; Johnson and Larsson, 1985; Johnson and Rath, 1991; Rath et al., 1996;

Johnson, 1998; Foote, 1999; Johnson and Guthrie, 2000). Many modifications of extenders

and freezing procedures were developed (see Johnson, 1998), including the pellet method

of freezing, which was developed originally in Japan (Nagase and Graham, 1964).

Pregnancy rates and litter sizes are reduced with cryopreserved boar sperm, so frozen

semen is limited to use in special breeding programs. Fresh or extended liquid semen is

used for about 99% of AI in swine. Rapid transport of extended semen makes it feasible for

swine farmers to take advantage of commercially produced boar semen. Large

corporations increase the services possible from their own boars by using AI. Insemination

is done by the swine farm personnel. A typical dose is 3 × 109 sperm inseminated in 80 mL.

Over 50% of the sows in the United States are inseminated today, and about 80% farrow

with 10 pigs per litter. Swine AI is growing at a phenomenal rate. Techniques for evaluating

quality of boar sperm are similar to those used for bull sperm (Johnson, 1998). Sexing of
boar sperm is possible but is too slow to produce sexed sperm for commercial use.

Detection of estrus is facilitated in sows because most come into estrus within a week after

weaning their litters. Further technical details can be found in the references cited

previously.

Artificial insemination for Horse

Research on AI of horses started in Russia in 1899 (Ivanow, 1907; Ivanoff, 1922),

stimulated particularly by the military’s need for horses. Ishikawa initiated similar studies

in Japan in 1912 (Sato, 1916; Nishikawa, 1962). The book by Nishikawa provides a

fascinating account of the extensive, detailed studies by a great researcher and teacher and

an extraordinary gentleman. After visiting in our home in the 1960s he wrote that he

couldn’t wait for me to visit him in Japan where he could “hospitalize” me. I was treated

royally. McKenzie et al. (1939) and Berliner (1942) initiated studies on the collection,

processing, and insemination of stallion and jack semen in the United States. The earliest

collections of semen were obtained by placing a rubber semen collection bag in the vagina

of a mare in estrus. In the 1930s and 1940s, several types of artificial vaginas were

developed (Anderson, 1945; Maule, 1962; Perry, 1968; Davies Morel, 1999) and they since

have been modified. A smaller, lighter version was developed by Nishikawa (see Davies

Morel, 1999). Semen evaluation is performed similarly to the evaluation of bull semen.

Military studs provided a convenient source of stallions (Anderson, 1945; Maule, 1962),

but after World War II many of these were eliminated because the horse population had

declined. In addition, the restrictive regulations on the use of AI by several equine breed
organizations inhibited research and the application of AI. China was the major country

using equine AI during this period. Advances made in cryopreserving bovine sperm

stimulated interest in cryopreservation of equine semen. Research results are summarized

in several congresses and symposia (Nishikawa, 1964, 1972; Bonadonna and Succi, 1976;

Sharp and Bazer, 1995) and by Davies Morel (1999). Although methods have been devised

to freeze stallion sperm, most equine AI is done with cooled, extended semen used within

48 h of collection during the spring breeding season. The breeding season may be advanced

by fall lighting.

Artificial insemination for Sheep and Goat

The early development of AI in sheep on a major scale began in Russia (Milovanov,

1938, 1964; Maule, 1962), where the collective farms provided an ideal arrangement for

establishing AI programs. China also has extensive sheep AI programs. Artificial

insemination spread to central Europe and also was widely applied commercially in France

and Brazil (Anderson, 1945; Maule, 1962; Foote, 1999). The techniques for semen

collection and artificial insemination in sheep and goats have been described in detail

(Evans and Maxwell, 1987). Semen quality and breeding efficiency are affected by season.

Both rams and bucks can be trained to serve the artificial vagina. However, for obtaining

semen from a large number of rams in the field, electroejaculation is a useful procedure,

pioneered by Gunn (1936) and applied to many species (Dziuk et al., 1954). Much of the

early research in the Western world on extenders for sperm, freezing of semen, and AI

techniques was done by Emmens and Blackshaw, followed by Salamon and Maxwell in
Australia, and Dauzier, Colas, and Cortell in France (Corteel, 1981; Salamon and Maxwell,

1995a,b; Maxwell et al., 1999). Buck sperm cryopreservation is more successful than the

cryopreservation of ram sperm. The techniques and media for freezing semen such as with

egg yolk-trisglycerol were modified (Corteel, 1981; Salamon and Maxwell, 1995a; Maxwell

and Watson, 1996; Amoah and Gelaye, 1997) from procedures developed for bull sperm

(Davis et al., 1963). Frozen-thawed semen results in satisfactory fertility in goats provided

that the sperm are deposited deep into or through the cervix. In the ewe this is difficult.

Therefore, insemination into the uterus with the aid of a laparoscope has been necessary to

achieve high fertility. Recently, Maxwell et al. (1999) used intracervical insemination

successfully by adding seminal plasma to cryopreserved ram sperm before it was used for

insemination. Because of the difficulty of insemination, general management and low value

per animal, AI, particularly of sheep, is not widespread.

Artificial insemination for Poultry

Artificial insemination has been widely applied to poultry. Semen collection,

processing, and AI have been reviewed by Sexton (1979) and Lake (1986) and more

recently by Donoghue and Wishart (2000). Pioneers in the poultry field were Burrows and

Quinn (1937), who developed the method of abdominal massage and pressure to collect

semen. With the ease of collecting poultry semen, and proximity of hens on large breeding

farms, AI is used extensively with freshly collected semen. It is used 100% for turkey

breeding because mating is difficult. Freshly collected chicken semen was among the first

type of semen to be frozen (Shaffner et al., 1941; Polge et al., 1949). However,
cryopreserved poultry sperm are less fertile and freezing poultry sperm still is

experimental (Gill et al., 1999).

Artificial insemination for other Domestic Mammals

Although the dog was the first animal in which AI was documented, AI has only been

used in special cases, such as for breeding guide dogs or for overcoming special problems.

Many breed organizations do not register puppies produced by AI. Cryopreservation of dog

sperm is successful, using modifications of the yolk-tris and yolk-lactose extenders

developed for cryopreserving bull sperm (Foote, 1990). Rabbits have been extensively

used as a model for large animals and humans (Foote, 1998; Foote and Carney, 2000). All

the reproductive techniques employed with farm animals can be performed with the low-

cost rabbit model, and certain placental membrane characteristics make them especially

relevant for studies of human teratology (Foote and Carney, 2000).

Artificial insemination for other endangered mammals

The first animal reproductive biotechnology used to preserve endangered species

was AI (Wildt et al., 1995). Again, many of the principles and procedures used were

adapted from cattle (Watson, 1978).

 Relationship of frozen-thawed semen quality with the fertility rate after being
distributed in the Brahman Cross Breeding Program

(Berlin Pandapotan Pardede, Muhammad Agil, Yudi Yudi, and Iman Supriatna)

Various factors can reduce the quality of semen used for artificial insemination and

have an impact on fertility decline, such as poor handling during frozen semen distribution.

This study was aimed at assessing the quality of frozen-thawed semen after distribution in

the field and its importance in maintaining fertility. The Brahman Cross (BX) breeding

program of PT Lembu Jantan Perkasa, Indonesia, was used. This program was preferred

due to its adherence to guidelines that limit the effects of extraneous factors that may affect

semen quality. Frozen-thawed semen samples from eight bulls with the same production

code were analyzed and compared between the production site (artificial insemination [AI]

center) and the field (BX breeding program). Total and progressive motility (PM) of sperm

were determined using computer-assisted semen analysis. Plasma membrane integrity

(PMI) was assessed using hypo osmotic swelling test, sperm viability using Eosin-Nigrosin

staining, acrosome integrity using trypan blue-Giemsa staining, morphological

abnormalities using William staining, and DNA fragmentation using toluidine blue staining.

The fertility rate was determined using the conception rate (%) derived from AI data based

on 502 AI services and478 cows in the BX breeding program. A t-test was used to compare

the quality of frozen-thawed semen before and after distribution. The relationship between

the qualities of frozen semen after distribution in the field with fertility was analyzed using

Pearson correlation. There was no significant difference (p>0.05) in the quality of frozen-

thawed semen (sperm motility, PMI, viability, acrosome integrity, abnormalities, and DNA

fragmentation) between the production site (AI center) and after distribution in the field
(BX breeding program). The semen met the minimum standards for AI programs. Total

motility (r=0.986),PM (r=0.961), sperm viability (r=0.971), PMI (r=0.986), and acrosome

integrity (r=0.992) were all positively correlated (p<0.05) with fertility rate; while sperm

abnormalities (r=−0.996) and sperm DNA fragmentation (r=0.975) were negatively

correlated (p<0.05) with fertility rate. The study showed that to achieve the maximal and

optimal fertility rate in bulls in an AI program, the overall quality of frozen-thawed semen

in all aspects is critical. This can be achieved if the handling during distribution and

storage,as well as the various factors that may affect the quality of semen in the field, can

be controlled properly.
 Factors affecting conception rate after the first artificial insemination in a private
dairy cattle farm in North Algeria

(Samir Souamesand Zahra Berrama)

This study analyzed risk factors influencing the conception rate at the first artificial

insemination (CR1) in dairy cows reared in the plain of Mitidja, which is considered a major

dairy region in North Algeria. A total of 1054 lactations were used in the relational study of

fertility conducted using the multivariable logistic regression model using the odds ratio.

The breeding season had a specific effect on fertility; the first service was often followed by

pregnancy when performed during autumn (AUT) (OR=1.67, p<0.05) and spring (SPR)

(OR=1.65, p<0.05). The chances of obtaining conception during the first service increased

significantly for a waiting period (WP) (interval between calving and time to first service)

of 50-100 days postpartum (DPP) (OR=1.67, p<0.05). From this study, it can be concluded

that no specific effect was observed relative to the breed and parity. Furthermore, CR1

significantly increased after summer calving when the first services were performed during

SPR and AUT and a WP after 50 DPP.

 Comparative analysis of various step-dilution techniques on the qualityof frozen
Limousin bull semen

(Ani Atul Arif, Tulus Maulana, Ekayanti Mulyawati Kaiin, Bambang Purwantara, Raden Iis
Arifiantini, andErdogan Memili)

Indonesia has two National Artificial Insemination centers and 17 Regional Artificial

Insemination Centers. The frozen semen production techniques differed between the

centers, including the type of diluent and semen dilution technique. The aim of the research

was to compare the quality of frozen Limousin bull semen diluted using different

techniques. Semen was collected from three sexually mature Limousin bulls using an

artificial vagina. Immediately after collection, the semen was evaluated macroscopically

and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality

was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three

different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room

temperature ([RT] 20°Cuntil 25°C) two-step dilution (50% SMEY without glycerol at RT,

stored at 5°C; and 50% SMEY with 16% glycerol after1 h stored at 5°C); and three-step

dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with

16%glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was

loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm

motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde

(MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing. The

results showed that there were no significant differences in sperm motility and DNA

integrity between dilutions (p>0.05). However, sperm viability and membrane intactness

of one-step dilutions were higher than those of three-step dilutions. The concentrations of
MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step

dilutions (p<0.05). It was concluded that the one-step-dilution technique was better than

three-step dilution for cryopreservation of Limousin bull semen.

 Utilization of bull fertility-associated antigen to improve the quality offrozen bull
semen

(Tri Wahyu Suprayogi, Hardijanto Hardijanto, Mas’ud Hariadi, Fedik Abdul Rantam
and Win Darmanto)

The implementation of artificial insemination (AI) is one of the strategies to use

superior male semen optimally to improve the genetic quality of livestock. One of the

factors that influence AI is a fertility-associated antigen (FAA). This research aimed to

examine the effects of FAA extracted from the accessory sex glands of a bull from a

slaughterhouse that was added in bull semen freezing medium to increase cattle (bull)

fertilization. This research used a randomized complete block design. It consisted of two

research phases, namely, explorative and experimental phases. The first phase involved

determining the FAA molecular weight using the SDS-PAGE method, and the second phase

consisted of laboratory and field testing, including testing the quality of frozen semen

supplemented with FAA extracted from the accessory glands of a bull’s genital organ from a

slaughterhouse with various doses (0, 5, 10, and 15 µg in every 200 million progressively

motile spermatozoa). The results showed that the percentages of bull sperm motility

between the groups without and with the additional administration of FAA with a dose of 5

µg did not significantly differ. However, there was a difference between the groups without

and with the additional administration of FAA with doses of 10 and 15 µg. After further

testing, the highest percentage of sperm progressive motility occurred at a dose of 15

µg/200 million progressively motile spermatozoa (P3),which was equal to 2.59±46.88b

(%). This research found that not all of the accessory glands (seminal vesicles) of bulls

taken from the slaughter house contain the FAA. An FAA level between the accessory
glands (seminal vesicles) of one cattle to another is different. The addition of the FAA

protein from the accessory sex glands of a bull’s organ in cattle semen can improve fertility

by increasing the percentage of viability, motility, intact plasma membrane of spermatozoa,

and pregnancy rate of bulls and decreasing the sperm capacitation post-thawing.
 Influence of bull age, ejaculate number, and season of collection on semen
production and sperm motility parameters in Holstein Friesian bulls in a commercial
artificial insemination centre

(Edel M Murphy, Alan K Kelly, Ciara O’Meara, Bernard Eivers, Patrick

Lonergan, and Sean Fair)

In the current era of genomic selection, there is an increased demand to collect

semen from genomically selected sires at a young age. The objective of this study was to

assess the effect of bull age, ejaculate number, and season of collection on semen

production (ejaculate volume, sperm concentration, and total sperm number; TSN) and

sperm motility (pre-freeze and post-thaw total and gross motility) parameters in Holstein

Friesian bulls in a commercial artificial insemination (AI) center. The study involved the

interrogation of a large dataset collected over a 4-yr period, (n = 8,983 ejaculates; n = 176

Holstein Friesian bulls aged between 9 mo and 8 yr). Bulls aged less than 1 yr had the

poorest semen production and sperm motility values for all parameters assessed compared

with bulls older than 1 yr (P < 0.01). First ejaculates had greater semen production and

greater pre-freeze motility values than second consecutive ejaculates (P < 0.01), but

despite this, there was no difference in post-thaw motility. When subsequent ejaculates

were collected from bulls aged less than 1 yr, semen production and sperm motility did not

differ compared with mature bulls. Semen collected in winter was poorest in terms of

sperm concentration and TSN, but best in terms of post-thaw motility (P < 0.01). In

conclusion, second ejaculates can be collected, particularly from bulls aged less than 1 yr,

without a significant decrease in post-thaw sperm motility, thus may be a useful strategy to

increase semen availability from young genomically selected AI bulls in high demand.
 Individual variation in fresh and frozen semen of Bali bulls (Bos sondaicus)

(R. Indriastuti, M. F. Ulum, R. I. Arifiantini and B. Purwantara)

Bali cattle are a native Indonesian breed that has many advantages over other cattle.

They easily to adapt to various types of feed, environmental conditions, and extreme

climate changes, and they have good reproductive efficiency. This study aimed to analyze

the individual factors influencing the sperm quality of Bali bulls at Baturiti Artificial

Insemination (AI) center. Semen that was ejaculated from nine Bali bulls was collected

using artificial vaginas (n=5/bull).Semen ejaculates were evaluated immediately after

collection to measure the quality of the fresh semen, including semen volume, sperm

concentration, progressive motility, membrane integrity (MI), and abnormal morphology.

Frozen semen was evaluated for progressive sperm motility, concentration, viability, MI,

abnormal morphology, and deoxyribonucleicacid (DNA) fragmentation. Other secondary

data, focusing on semen quantity (semen volume and sperm concentration),were also

collected from frozen the semen production data of the Baturiti AI center from 2017 to

2019. Data were analyzed statistically using a completely randomized design, and one-way

analysis of variance was applied to find differences among individual bulls. Significant

differences (p<0.05) were found among the bulls in semen volume, sperm motility,

concentration, and MI of the fresh semen. Significant differences (p<0.05) were also found

among the bulls in sperm motility, viability, MI, abnormal morphology, and DNA

fragmentation of the frozen semen. Individual variation in all the tested sperm parameters

of the fresh semen of Bali bulls, except sperm viability and abnormalities, was noted.
Similarly, individual variation in all the tested sperm parameters in frozen semen, except

sperm concentration, was noted. Therefore, individual factors can be used for selecting a

superior bull in Bali cattle.

 Pregnancy rates in hair sheep after Ovsynch synchronization and acombined
intracervical fixed-time artificial insemination and 10-daymating period

(D. A. Vallejo, J. D. Londoño, Y. A. Yepes, V. Tamayo, A. F. Mejia and J. G. Maldonado)

Pregnancy rates in hair ewes using an Ovsynch synchronization protocol under a

breeding system that combines fixed-time insemination plus a 10-day mating period as an

alternative. Ewes were fixed-time inseminated (fixed-time artificial insemination [FTAI])

with fresh semen, collected just before the insemination time through vaginoscopy at 16 h

after the second GnRH (gonadorelin) injection. Each experimental group was placed

separately during 15 days and, after this time, fertile rams were allowed back with ewes for

a 10-day mating period. Control group ewes remained with the rest of the herd suitable for

breeding and were bred under NM. Pregnancy diagnosis was performed by ultrasound

at28-, 56-, and 84-day post-breeding to differentiate between FTAI and NM pregnancies.

Total (FTAI±NM) pregnancy rates at 56-day post-breeding were used to compared Pre-

synch, Ovsynch, and control. For this purpose, two-tailed proportions comparison z-test

was used with a 95% confidence level, for testing as the null hypothesis whether two

proportions were equal. Pregnancy rates were higher in control ewes (66.4%) than FTAI

(46.6%). When pregnancy rates after a 10 day mating period (40%) were added, the final

rate (86.6%) was significantly (p<0.05) higher in Ovsynch-based protocols. The pregnancy

rate was significantly lower in FTAI ewes compared to FTAI +10-day mating group

(p<0.05). The overall pregnancy rate was 88.0, 85.7, and 67.0 (p>0.05) for Pre-synch,

Ovsynch, and control ewes, respectively.

 CONCLUSION

The opportunity to use artificial insemination (AI) is advantageous in the animal

industry, as it significantly speeds up the progression of genetic merit and synchronizing

the lactation period of groups of dams that can later be managed easily. This technology

also dramatically affect particularly in the beef cattle industry. Currently, about six percent

of all beef cattle producers use AI and/or estrus synchronization in their beef herds.

Artificial insemination seems to have a practical application to range cattle production as

shown at approximate 80 percent calf crop secured by these methods as compared to

about 40 to 65 percent calf crop secured under natural breeding conditions. In addition,

One of the primary deterrents to beef cattle producer adoption of AI is the perceived cost

required for breeding and maintenance expenditures.

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