Reproduction in Domestic Animals

Sanitary Procedures for Extended Semen 375Fig. 1. Phases of bacterial growth (adapted from Banwart 1979)geometric progression in cell number. Essential componentsof this growth process may include an increase incell mass and ribosome number, duplication of thebacterial chromosome, cell wall and plasma membranesynthesis, chromosomal partitioning with septum formation,and finally cell division. This rapid growthphase eventually reaches a point at which it plateaus,transitioning into the stationary phase. The bacterialconcentration and length of time of the stationary phasehas traditionally been thought to be controlled bynutrient exhaustion, physical space limitation, and ⁄ oraccumulation of inhibitory levels of metabolites, includingend products. Recent work has also alluded to thesecretion of quorum-sensing compounds, which allowfor interbacterial communication in the gauging ofpopulation density (Carbonell et al. 2002; Gonzalez andKeshavan 2006; Fulghesu et al. 2007). If no change ismade in the present conditions, a death phase eventuallycommences, which is driven by the decline in the viablecell population.As with spermatozoa, bacteria are unable to regulatetheir temperature. Spermatozoa are dependent upon theenvironment to provide this critical component toprolonged viability. Likewise, this component is ofequal importance to the viability and growth of bacteria.All bacteria have an optimum temperature (T o ) – thetemperature at which maximum growth rate is attained.If the bacteria are exposed to environmental temperaturesabove their T o , plasma membrane fluidityincreases, with concomitant alteration of cell functionand decreased growth. At some temperature point, nofurther compensation can occur, leading to perturbationof the cell and death. Spermatozoa are much moresensitive to increased temperature change than manyother cell types, including bacteria. Unlike bacteria,sperm do not have intracellular organelles to aid inmodifying plasma membrane fluidity for survivability.Consequently, exposure to a matter of a few degreesabove body temperature is all that is necessary for spermdeath to occur.As environmental temperature decreases (e.g. chillingfresh-extended semen), cell plasma membrane fluiditychanges in conjunction with decreased growth rate andmetabolism. At some point, growth and metabolismcease, and the cells become dormant. This phenomenonis employed to reduce metabolism and induce dormancyof sperm in extended semen. Although this is beneficialto sperm longevity, it can benefit contaminant bacteriaeven more, as they have the intracellular machinery toadapt to lower environmental temperatures to the pointthat some genera may again thrive giving way to theirprofuse overgrowth in the chilled extender.From prior studies in swine, the majority of contaminantsidentified in extended semen are Gram-negativebacteria, with a majority of the contaminant bacteriabelonging to the Family Enterobacteriaceae (Althouseet al. 2000, 2008; Althouse and Lu 2005). Our laboratoryhas seen similar types of contaminants in extendedsemen from other species as well. In these and otherstudies, bacterial contaminant overgrowth of certaingenera has been associated with deleterious effects onsemen quality and longevity (Sone et al. 1989; Althouseet al. 2000; Akhter et al. 2008; Aurich and Spergser2007). Spermatozoal agglutination, decreased spermatozoalmotility and viability are potential end effects tobacteriospermic overgrowth. When such doses are usedin an AI programme, increased regular returns tooestrus, post-insemination vulvar discharges andreduced herd reproductive performance have beenreported in some species (Sone et al. 1989; Althouseet al. 2000; Bukharin et al. 2000). Depending uponbacterial type and species along with species origin ofthe sperm, an actual sperm to bacteria (CFU) ratio ofapproximately 1:1 up to 100:1 in a sample appearssufficient to generate the undesired effects on the semendose and fertility (Tamuli et al. 1984; Auroux et al.1991; Althouse et al. 2000), with these effects being of atime-dependent nature. Hence, extenders which providethe ability to extend storage life of sperm also providethe opportunity for certain contaminant bacteria toreach their necessary threshold for eliciting a negativeeffect on fertility potential.Sources and Control of ContaminationUsing pigs as a model, multiple contamination pointshave been identified in the production of extendedsemen (Althouse et al. 2000; Althouse and Lu 2005).From this work, contamination sources have beenclassified into either being of mammalian or nonmammalianorigin, and are usually found to originatefrom a single source on a stud ⁄ farm. These singlesources have been found to yield the same or differentbacterial strains at different times at the stud ⁄ farm.Likewise, different sources of contamination have beenfound at different times on the same stud ⁄ farm. Commonsources of bacterial contamination of extendedsemen are outlined in Table 1. Many of these sources,once contaminated, act as seeding points for furtherejaculates which come into contact with this source.From our laboratories work performed to date,a multitude of contamination sources appear to exist.The first step in minimizing bacterial contaminationof extended semen is the practice of good hygiene andgeneral sanitation by personnel. Personnel which comein contact with any materials which are subsequentlyused in the collection and processing of semen, or whoÓ 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag