ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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86Artificial Insemination in Farm Animalsdistribution of cholesterol within the membrane render boar spermatozoa very susceptibleto cold temperatures resulting in increased permeability and loss of controlled membraneprocesses (De Leeuw et al., 1990). Hence, rapid cooling of ejaculates to 15°C or coolingbelow 15°C results in loss of viability or cold shock (Johnson et al., 2000). To avoid this coldshock, prediluted ejaculates are better left at temperatures above 15°C for several hours toinduce cold resistance. In practice, semen is collected in isolated cans to avoid contact withcolder surfaces and subsequent dilution is done in a manner in which temperature isdiminished gradually. Two different protocols are normally used for this purpose: 1) Onestep dilution with either preheated diluter (~33°C) or diluter at room temperature or 2) atwo steps dilution with first a 1:1 dilution with preheated diluter (~33°C), followed by asecond dilution in either a preheated diluter or a diluter kept at room temperature(Waberski, 2009). After the final dilution, filling of commercial doses is done and the semenis allowed to cool down gradually to 17°C. When semen doses are to be transported, specialprecautions are taken to avoid temperature fluctuations (Green and Watson, 2002). Furtherstorage of diluted semen is done at 17°C, at which temperature semen metabolism isreduced (Althouse et al., 1998), a condition necessary to extend storage time.4.2 Storage mediumThe storage media for liquid boar semen aim to prolong sperm survival, to provide energyto the cells, to buffer the pH of the suspension and to avoid the growth of bacteria (Vyt et al.,2004a). Therefore, porcine semen extenders contain ions to maintain the osmotic pressure ofthe medium, glucose as energy source, buffering systems to stabilize the pH of the extenderand EDTA and antibiotics to prevent bacterial overgrowth (Johnson et al., 2000). Thepresence of glucose as the only energy source and the low oxygen content in the recipient inwhich diluted semen is stored stimulates the glycolytic metabolism. Consequently, theintracellular pH of spermatozoa is lowered which reduces their motility and enables them tosurvive several days at ambient temperature (Henning et al., 2009). Glucose also contributeslargely to the osmotic equilibrium. The ions in the media for liquid boar semen are merelysodium bicarbonate and sodium citrate and are simultaneously used as buffer. In BTSextender, also KCl is added to prevent the potassium loss from inside the cells, andsubsequent loss of motility due to Na-K pump inefficacy. Porcine spermatozoa are rathertolerant to minor changes in osmolality of the extender (Johnson et al., 2000). Iso-osmoticand slightly hyper-osmotic media are preferred for optimal preservation of fertilizingcapacity (Weitze, 1990). Incubation in media below 250 mOsm and above 300 mOsmrendered irreversible damage to the membranes and subsequent loss of motility.EDTA is added for its chelating properties. When Ca-ions are captured, the initiation ofcapacitation is inhibited (Watson, 1995). As a consequence, the fertilizing capacity of thespermatozoa is preserved. Depending on the composition of the extender, semen can bestored for 2 to 3 days in short-term extenders and up to five days or longer in long-termextenders (Johnson et al., 2000). Long-term extenders differ from short-term extendersmainly by the use of complex buffering systems (HEPES, Tris), mostly in addition to thebicarbonate buffering system, and by the presence of Bovine Serum Albumin (BSA). Thelatter has a positive influence on sperm survival due to the absorption of metabolic bacterialproducts from the extender. Cysteine is used as a membrane stabilizer (Johnson et al., 2000)inhibiting capacitation.To prevent bacterial proliferation during storage, antibiotics are added to the extender.Bacteria originate mostly from the prepuce, thus depending on the semen collection
Artificial Insemination in Pigs 87technique, from semen manipulation or from the water used in the extender preparation(Althouse and Lu, 2005). Depending on the species, bacteria have deleterious effects onsemen quality, namely depressed motility, cell death and agglutination (Althouse al., 2000),either by direct effect on the spermatozoa or by acidifying the environment. Europeanlegislation prescribes an antibiotic combination equivalent to 500 IU/ml penicillin, 500IU/ml streptomycin, 150 mg/ml lincomycin and 300 mg/ml spectinomycin, for having abroad antibacterial spectrum and activity towards leptospira. In practice most commercialextenders use aminoglycosides, especially gentamycin (Althouse and Lu, 2005; Vyt et al.,2007). However, bacterial contamination should be first minimized by good hygiene andgeneral sanitation by personnel (Althouse, 2008).The extender-concentrates are diluted in distilled or de-ionized water. Next to the bacterialquality of the water, the electrolyte content, especially the absence of calcium ions, is animportant characteristic for the water used to make the extender.The comparison of different semen extenders has been subject of two kinds of studies:studies comparing different extenders in vitro, focusing on quality of semen after storage(De Ambrogi et al., 2006; Vyt et al., 2004a) and studies comparing fertility in vivo afterinsemination of semen stored for several days or stored in different extenders (Haugan al.,2007; Kuster and Althouse, 1999).In vitro experiments showed no differences in cell viability between short-term and longtermextenders during 9-day storage (De Ambrogi et al., 2006). Motility remainedunchanged within the first 72 hours, even in BTS-extenders, the most widely used shorttermextender. Based on in vivo results, differences were noticed between different extendersbut it was not always possible to relate these differences to the type of extender. Differencesbetween long-term extenders were observed from day 4 of storage onwards (Kuster andAlthouse, 1999). The limited number of extenders compared in each study makes it difficultto set up a ranking of the semen preserving quality of long-term semen extenders.4.3 Storage of porcine semen in frozen stateAs mentioned above, porcine spermatozoa are particularly sensible to low temperatures andto rapid cooling due to the specific composition of the cell membrane (De Leeuw al., 1990).Cold shock can be solved technically by inducing cold resistance, namely incubating spermat ambient temperature for several hours (Watson, 1995), by contact with seminal plasmathat has a protective effect on spermatozoa (Centurion et al., 2006), together with controlledfreezing protocols. The variation in freezability of individual boar’s semen is however moredifficult to solve.Semen extenders for frozen boar semen are completely different from extenders for liquidsemen. The presence of egg-yolk, containing low density lipoproteins and cholesterol, has aprotective effect on sperm membrane during cooling (Bathgate et al., 2006). Cryoprotectants,especially glycerol are added in low concentration to the medium in order to diminishmembrane damage by freezing. Additionally, sugars and synthetic detergents are added,the latter having a modifying effect on the egg yolk inducing a better membrane stability ofthe cell membrane (Johnson et al., 2000). Thawing of the semen dose has been another pointof concern. Thawing has to be fast in order to maintain sperm motility and acrosomeintegrity afterwards. Both processes i.e. freezing and thawing result in plasma membranechanges, explaining the variety of protocols available.The fertility results with frozen semen have improved: cervical insemination results in a75% farrowing rate and a litter size of 9.6 (Roca et al., 2003). Nevertheless, freezing and
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VIContentsChapter 9Evaluation of a
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