**3.2. Sperm concentration**

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

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

Primarily due to the anatomical variation of the phallic region in different birds, semen collection techniques will vary. In contrast to ratites and water-fowl with an intromittent phallus, Galliformes (chicken, turkey, and quail) do not have an intermittent organ. Their nonintromittent organ consists of folds and bulges that make contact with the female's cloaca at mating. From an anatomical perspective, there are considerable differences between the nonintromittent organs of the chicken and turkey (Figure 6). The rooster has a prominent medial phallic body and relatively small lateral phallic bodies and lymph folds. Conversely, the turkey tom has no medial phallic body but prominent lateral phallic bodies and lymph folds. Sex sorting at hatch by cloacal examination is based on the relative differences in size of these

**Figure 6.** The turkey (left) and chicken (right) cloacae are viewed with the dorsal lips of the cloacae pulled back to expose each species phallus non-protrudens. Unlike the turkey, the chicken has a central protuberance, the medial phallic body (MPB) and regressed lateral phallic bodies (LPB) and lymph folds (LF). The turkey phallus non-protrudens

The goal of semen collector is to obtain the maximum volume of clean, high quality semen with the minimal amount of handling. In chickens and turkeys, the abdominal massage technique [1, 4] involves massaging the cloacal region to achieve phallic tumescence. This is followed by a 'cloacal stroke', a squeezing of the region surrounding the sides of the cloaca to express the semen. Little additional semen can be expressed after two cloacal strokes; addi‐ tional cloacal strokes may cause damage to the phallic and cloacal regions and contribute to

is characterized by dominant LPB and LF and the conspicuous absence of the MPB.

semen contamination [85].

technology and reproductive biology for ratites were reviewed by Malecki et al. [84].

184 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

**3.1. Semen collection**

structures between the males and females.

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 spermatocrit) and optical density (OD; photometry).

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 concentration and the derivation of standard curves.)

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.

Another concern when using any semen evaluation method is variation in the operator's techniques. Consistency is the key to repeatable data. The technical staff all must follow the same standard operating procedures (SOPs). For example, when counting sperm with a hemocytometer, all individuals in a lab should following the same SOP for how long the sperm are permitted to settle on the grid and which sperm to count or omit from the count. Also, is the photometer zeroed with the same buffer? If a procedure calls for an incubation period, such as in a live-dead stain, are the samples being incubated for the same duration each time using the same stain concentrations? A lack of consistency in following the SOPs within a laboratory will lead to unwarranted variation and non-reproducible and inaccurate data.

**3.4. Sperm motility and mobility**

viscous medium (Accudenz) at 41°

**3.5. Evaluation of fertility**

accurate and consistent preparation of the reagents.

The advantages here are speed and the PL remains wrinkle free.

Sperm motility can be progressive (forward direction) or non-progressive (random movement or oscillations) movement. Generally, progressive motility is determined subjectively at ambient temperature using a microscope at low magnification (hanging-drop technique) or objectively using a computer-assisted semen analysis system. These techniques are reviewed by Bakst and Long [4]. Motility evaluated by microscopy has been shown to have little correlation with fertility and simply reveals that the sperm are motile. First described by Froman and McLean [87] and further elaborated for commercial use by Froman [88], the sperm mobility assay has gained popularity as a measure of an individual male's ability to produce highly mobile sperm [mobility defines the ability of sperm to move progressively against a

producing less mobile sperm. While the sperm mobility assay is a powerful tool for the selection of the most fecund males to be used in AI, it necessitates attention to details and

The measure of a successful AI program is sustained hen fertility. While candling-fertility is useful, there is an eight or more day lag between the last AI and candling-fertility determina‐ tion, which overlaps with the next insemination (hen insemination is generally at 7-day intervals). With AI programs, it is often desirable to determine the fertility status of a flock before the next weekly insemination. There are several options available: breaking-out fresh eggs and examining the GD to differentiate a fertilized from an unfertilized or early dead embryo; setting normal but culled eggs (checked, hairline cracked, or dirty eggs) in a spare incubator for 24-36 hr before breaking-out [89]; counting sperm in the outer PL; and counting sperm holes in the inner PL. The above procedures are reviewed in Bakst and Long [4].

As noted previously, the sperm penetration assay is not only used to determine true fertility, but also to estimate the number of sperm residing in the use SSTs at the time of ovulation [90]. The isolation of the inner PL and staining procedure, initially developed for chicken eggs by Bramwell et al. [91], was quickly adapted to turkey eggs by Donoghue [92]. The major drawback to the sperm penetration assay as originally described is that it is time consuming, particularly with respect to isolating, washing, and positioning the PL wrinkle-free on the slide. Spasojevic [10] and colleagues at Willmar Poultry Company (Willmar, MN) significantly increased the efficiency of preparing the PL slides from turkey eggs in the following manner: the albumen is removed from the ovum as in the original procedure [4]; a square is outlined on a slide using super glue; the slide is placed firmly on the ovum's surface with the GD centered in the square; after the glue is set, the PL is cut and washed to remove adhering yolk.

A different modification of the sperm penetration assay was suggested by I.A. Malecki (personal communication) and entails placing a filter ring over the GD (inside diameter slightly larger than the GD), cutting around the outside diameter of the filter ring (about 2 mm between the inside and outside perimeter of the ring), and lifting the filter ring off the ovum. The filter ring with the adhering PL is washed gently with saline to remove the yolk and GD material

C] that are more likely to fertilize an ovum than males

Artificial Insemination in Poultry http://dx.doi.org/10.5772/54918 187

### **3.3. Sperm viability**

In the context of semen evaluation, reference to 'viable' sperm simply implies that such sperm possess an intact plasmalemma and are assumed to be functional. Plasmalemma in‐ tegrity is frequently determined using either a dead-cell or a live-cell stain alone or simulta‐ neously. The dead-cell stains are excluded by sperm with an intact plasmalemma but stain dead sperm possessing a permeable plasmalemma. Live-cell stains permeate the intact sperm plasmalemma and become visible only after reacting with cytosolic enzymes or inter‐ acting with sperm nuclear proteins. Both eosin and propidium iodide are popular dead-cell stains while calcein AM and SYBR-14 are frequently used live-cell stains (see [86] for extend‐ ed discussion and availability for the live-cell probes). On a commercial breeder farm, the nigrosin/eosin (N/E) technique is most likely the procedure to be used to determine sperm viability [4]. Briefly, sperm are stained with N/E and a smear of the stained sperm is made on a slide (Figure 7). Under a bright field microscope the viable sperm remain pearly white, while eosin will stain non-viable sperm a pink to magenta color. The nigrosin serves as a background to enhance differentiation between the non-viable and viable sperm. In contrast to the N/E technique, a more sophisticated laboratory may use flow cytometry that sorts via‐ ble from non-viable sperm after staining with calcein AM or SYBR-14 and propidium iodide.

**Figure 7.** The left panel shows a nigrosin eosin preparation of turkey sperm with nearly 100% viable sperm (un‐ stained) white nuclei and midpieces. The sperm head is clearly visible as the white arcing segment; the acrosome and midpiece are difficult to differentiate from the nucleus. The upper right panel reveals a normal sperm and a second sperm with an abnormally curved and swollen midpiece. Observed in the lower right panel is a nonviable sperm stained with eosin throughout the nucleus and midpiece. Barely visible at the anterior end of the nucleus is the un‐ stained, conical shaped acrosome.
