ARTIFICIAL INSEMINATION

Artificial insemination, or AI, is the process by which sperm is placed into the reproductive tract of a female for the purpose of impregnating the female by using means other than sexual intercourse or NI.

Artificial insemination is widely used for livestock breeding, especially for dairy cattle and pigs. Techniques developed for livestock have been adapted for use in humans.

Specifically, freshly ejaculated sperm, or sperm which has been frozen and thawed, is placed in the cervix (intracervical insemination – ICI) or, after washing, into the female's uterus (intrauterine insemination – IUI) by artificial means.

Artificial insemination is used in many non-human animals, including sheep, horses, cattle, pigs, dogs, pedigree animals generally, zoo animals, turkeys and even honeybees. It may be used for many reasons, including to allow a male to inseminate a much larger number of females, to allow use of genetic material from males separated by distance or time, to overcome physical breeding difficulties, to control the paternity of offspring, to synchronise births, to avoid injury incurred during natural mating, and to avoid the need to keep a male at all (such as for small numbers of females or in species whose fertile males may be difficult to manage).

Semen is collected, extended, then cooled or frozen. It can be used on site or shipped to the female's location. If frozen, the small plastic tube holding the semen is referred to as a straw. To allow the sperm to remain viable during the time before and after it is frozen, the semen is mixed with a solution containing glycerol or other cryoprotectants. An extender is a solution that allows the semen from a donor to impregnate more females by making insemination possible with fewer sperm. Antibiotics, such as streptomycin, are sometimes added to the sperm to control some bacterial venereal diseases. Before the actual insemination, estrus may be induced through the use of progestogen and another hormone (usually PMSG).

Artificial insemination of farm animals is very common in today's agriculture industry in the developed world, especially for breeding dairy cattle (75% of all inseminations[clarification needed]) and swine (up to 85% of all inseminations). It provides an economical means for a livestock breeder to improve their herds utilizing males having very desirable traits.

Although common with cattle and swine, AI is not as widely practised in the breeding of horses. A small number of equine associations in North America only accept horses that have been conceived by "natural cover" or "natural service" – the actual physical mating of a mare to a stallion. The Jockey Club being the most notable of these - no AI is allowed in Thoroughbred breeding. Other registries such as the AQHA and warmblood registries allow registration of foals created through AI, and the process is widely used allowing the breeding of mares to stallions not resident at the same facility - or even in the same country - through the use of transported frozen or cooled semen.

In 1997, Tilikum, an Orca at SeaWorld Orlando began training for AI. In early 2000, Kasatka who resides at SeaWorld San Diego was artificially inseminated using his sperm. She gave birth to Tillikum's son, Nakai, on September 1, 2001. On May 3, 2002, another female in San Diego, named Takara, bore Tilikum's calf through AI, a female named Kohana. Takara is Kasatka's oldest child and daughter.

ANTIBIOTIC RESISTANCE MECHANISMS

Antibiotic resistance can be categorized in three types:

1. Natural or intrinsic resistance

• Inaccessibility of the target (i.e. impermeability resistance due to the absence of an adequate transporter: aminoglycoside resistance in strict anaerobes)
• Multidrug efflux systems: i.e. AcrE in E. coli, MexB in P. aeruginosa
• Drug inactivation: i.e. AmpC cephalosporinase in Klebsiella 

2. Mutational resistance

• Target site modification (i.e. Streptomycin resistance: mutations in rDNA genes (rpsL), ß-lactam resistance: change in PBPs (penicillin binding proteins))
• Reduced permeability or uptake
• Metabolic by-pass (i.e trimethoprim resistance: overproduction of DHF (dihydrofolate) reductase or thi^- mutants in S. aureus)
• Derepression of multidrug efflux systems

3. Extrachromosomal or acquired resistance (Disseminated by plasmids or transposons)

• Drug inactivation (i.e. aminoglycoside-modifying enzymes, ß-lactamases, chloramphenicol acetyltransferase)
• Efflux system (i.e. tetracycline efflux)
• Target site modification (i.e. methylation in the 23S component of the 50S ribosomal subunit: Erm methylases)
• Metabolic by-pass (i.e trimethoprim resistance: resistant DHF reductase)

RESISTANCE TO AMINOGLYCOSIDE ANTIBIOTICS

Aminoglycosides (Streptomycin, kanamycin, tobramycin, amikacin,...) are compounds that are characterized by the presense of an aminocyclitol ring linked to aminosugars in their structure. Their bactericidal activity is attributed to the irreversible binding to the ribosomes although their interaction with other cellular structures and metabolic processes has also been considered. They have a broad antimicrobial spectrum. They are active against aerobic and facultative aerobic Gram-negative bacilli and some Gram-positive bacteria of which staphylococci. Aminoglycosides are not active against anaerobes and rikettsia. Spectinomycin which is an aminocyclitol devoided of aminosugars is by extension included in the familiy of aminoglycosides. It also differs from them by its bacteriostatic ativity and by its way of action. Spectinomycin acts on protein synthesis during the mRNA-ribosome interaction and it does not lead to mistranslation like aminoglycosides do.

Three mechanisms of resistance have been recognized, namely ribosome alteration, decreased permeability, and inactivation of the drugs by aminoglycoside modifying enzymes. The latter mechanism is of most clinical importance since the genes encoding aminoglycoside modifying enzymes can be disseminated by plasmids or transposons.

Ribosome alteration

High level resistance to streptomycin and spectinomycin can result from single step mutations in chromosomal genes encoding ribosomal proteins: rpsL (or strA), rpsD (or ramA or sud2), rpsE (eps or spc or spcA). Mutations in strC (or strB) generate a low-level streptomycin resistance.

Decreased permeability

Absence of or alteration in the aminoglycoside transport system, inadequate membrane potential, modification in the LPS (lipopolysacchaccarides) phenotype can result in a cross resistance to all aminoglycosides.

Inactivation of aminoglycosides

These enzymes are classified into three major classes according to the type modification: AAC (acetyltransferases), ANT (nucleotidyltransferases or adenyltransferases), APH (phosphotransferases).

RESISTANCE TO TETRACYCLINE ANTIBIOTICS

Tetracyclines (tetracycline, doxycycline, minocycline, oxtetracycline, ...) are antibiotics which inhibit the bacterial growth by stopping protein synthesis. They have been widely used for the past forty years as therapeutic agent in human and veterinary medicine but also as growth promotor in animal husbandry. The emergence of bacterial resistances to these antibiotics has nowadays limited their use. Three different specific mechanisms of tetracycline resistance have been identified so far: tetracycline efflux, ribosome protection and tetracycline modification.

Tetracycline efflux is achieved by an export protein from the major facilitator superfamily (MFS). The export protein was shown to function as an electroneutral antiport system which catalyzes the exchange of tetracycline-divalent-metal-cation complex for a proton. In Gram-negative bacteria the export protein contains 12 TMS (transmembrane fragments) whereas in Gram-positive bacteria it displays 14 TMS. Ribosome protection is mediated by a soluble protein which shares homolgy with the GTPases participating in protein synthesis, namely EF-Tu and EF-G. The third mechanism involves a cytoplasmic protein that chemically modifies tetracycline. This reaction takes only place in the presence of oxygen and NADPH and does not function in the natural host (Bacteroides). 

The two first mechanisms are the most widespread and most of their genes are normally acquired via transferable plasmids and/or transposons. These two mechanisms were observed both in aerobic and anaerobic Gram-negative or Gram-positive bacteria demonstrating their wide distribution among the bacterial kingdom. To date, about sixty-one tetracycline resistance genes have been sequenced and thirty-two classes of genes identified in non-producers and producers (Streptomyces). Each new class is identified by its inability to hybridize with any of the known tet genes under stringent conditions (Levy et al. 1989. AAC 33:1373-1374). A new nomenclature for the resistance determinants has been proposed for the future with the S. B. Levy group to coordinate the naming of the detreminants (Levy et al. 1999. AAC 43:1523-1524).

RESISTANCE TO BETA-LACTAM ANTIBIOTICS

ß-lactams belong to a family of antibiotics which is characterized by a ß-lactam ring. Penicillins, cephalosporins, clavams (or oxapenams), cephamycins and carbapenems are members of this family. The integrity of the ß-lactam ring is necessary for the activity which results in the inactivation of a set of transpeptidases that catalyze the final cross-linking reactions of peptidoglycan synthesis.


In gram positive bacteria, especially staphylococcus aureus, resistance of penicillin G is mainly through the production of beta-lactamase enzymes that break the beta-lactam ring. S.aureus secretes beta-lactamse enzyme extracellularly as inducible exoenzymes that are plasmid-mediated. The inherent resistance to penicillin G of many gram negative bacteria result from low permeability of the gram negative cell wall, lack of PBP`s, and a wide variety of beta-lactamse enzymes. Most gram negative bacteria inherently express low levels of species-specific, chromosomally mediated beta-lactamase enzyme within the periplasmic space, which sometimes contribute to resistance. These enzymes hydrolyze susceptible cephalosphorins more rapidly than penicillin G, but they hydrolyze ampicillin, carbenicillin, and beta-lactamase-resistant penicillins poorly.

Production of plasmid-mediated beta-lactamase is widespread among common gram negative primary and opportunist bacterial pathogens. The enzymes are constitutively expressed, present in the periplasmic space, and cause high-level resistamce. The majority are penicillinases rather than cephalosphorinases. The most widespread are those classified on the basis of their hydrolytic activity as TEM-type beta-lactamases, which readily hydrolyze penicillin G and ampicillin rather than methicillin, cloxacillin, or carbenicillin. The less widespread OXA-type beta-lactamases hydrolyze penicillinase-stable penicillins (oxacillin, cloxacillin, and related drugs). Beta-lactamases probably evolved from PBP`s as a protective mechanism for soil organisms exposed to beta-lactamase in nature, in which they are thought to be widespread through their production by molds. Because of transferable resistance, beta-lactamase production by pathogens is now widespread.

A major advance has been the discovery of broad-spectrum beta-lactamase-inhibitory drugs (e.g. clavulanic acid, sulbactam, tazobactam). These drugs have weak antibacterial activity but show extraordinary synergism when administered with penicillin G, ampicilin, or amoxicillin because of irreversible binding to the beta-lactamase enzymes of resistant bacteria. Other beta-;actamase inhibitors, such as cefotaxime and carbapenems, have potent antibacterial activity in their own right.