**3.5. Evaluation of fertility**

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

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 until transparent, placed on a slide, and then fixed and stained with saline washes after each step. Our laboratory has used the filter ring technique with eggs from broilers, turkeys, ducks, and quail and it is now our preferred method for the performing the sperm penetration assay.

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