**3. Techniques in artificial insemination and fertility evaluation in poultry**

Semen should be pearly white, viscous, and clean. With each male collected, the semen collector should perform a visual examination of the semen at the time of ejaculation. This is easier with the turkey because the ejaculate accumulates on the phallus before it is collected by the 'milker' (semen collector). Off-color or watery semen, and semen contaminated with blood or fecal/ urates debris should not be used for insemination. Due to the increased volume of transparent fluid in rooster semen, which is a transudate derived from the phallus at the time of ejaculation,

Artificial Insemination in Poultry http://dx.doi.org/10.5772/54918 185

If semen is to be diluted, it is best .to have a known volume of semen diluent (a tissue culturelike medium formulated to sustain sperm viability) at ambient temperature in the semen receptacle before collection begins. For routine AI of turkey hens, semen from 10-12 toms are pooled in a single receptacle, mixing the semen gently after each male is collected. Semen volume is determined and if the AI dose is based on numbers of sperm (generally 250-350 million sperm per dose) sperm concentration is determined. The most popular techniques for determining sperm concentration are the packed cell volume (PCV; also referred to as a

Determining sperm concentration using PCVs is nearly identical to that of determining blood hematocrit values. Semen aspirated into micro-hematocrit tubes are centrifuged in a hema‐ tocrit centrifuge until the sperm are tightly packed (10 min); the percentage of packed sperm cells relative to the original semen volume in the micro-tube is determined. Sperm concentra‐ tion is derived using a conversion factor or standard curve previously derived by comparing and graphically plotting varying ascending sperm concentrations from hemocytometer counts to corresponding spermatocrit readings. (See [4] for detailed protocols to determine sperm

The optical density (OD) is determined using a photometer. The OD of highly diluted semen is directly proportional to the concentration of sperm, thus providing an indirect estimate of the sperm concentration. Like the PCV method, sperm concentration is derived using a conversion factor or previously derived standard curve by comparing and graphically plotting varying sperm concentrations from hemocytometer counts to corresponding OD readings [4]. The PVC and OD methods are two *indirect* methods of determining sperm concentration, that is, the final concentration is calculated from a regression equation or standard curve derived, in part, from *direct* sperm counts with a hemocytometer [4]. Briefly, to derive a regression equation and standard curve, serial dilutions (n=5) covering a wide range of sperm concen‐ trations are prepared and sperm concentrations are determined with a hemocytometer and the instrument or method that requires the standard curve (at least 4 replicates with 4 different semen samples). This is a tedious procedure but if reliable and repeatable sperm numbers are to be inseminated it is best to establish standard curves for each instrument every 12-18 months. The reason for this is that the rotational speed of different centrifuges and the intensity of a photometer's light source may differ as a result of manufacturer's variation, age of the instrument, and/or repeated use of the instrument, thereby producing variations in the

respective final readings and subsequent calculations of sperm concentrations.

chicken semen is less viscous and sperm concentration lower than that of turkey semen.

**3.2. Sperm concentration**

spermatocrit) and optical density (OD; photometry).

concentration and the derivation of standard curves.)

For non-domestic birds, chapters in Bakst and Long [4], Lake and Stewart [5] and Bakst and Wishart [6] provide overviews of semen evaluation and AI techniques. Artificial insemination technology and reproductive biology for ratites were reviewed by Malecki et al. [84].
