Variation in the number of spermatozoa in Blue Tit and Great Tit eggs

Auk, The, Jan 2000 by Lefjeld, Jan T, Aas, Christian K, Birkhead, Tim R, Fletcher, Fiona, Et al

In birds, fertilization takes place in the infundibulum at the top of the female reproductive tract. To reach this site, spermatozoa must travel from the hostile environment of the vagina, or from sperm-- storage tubules located at the utero-vaginal junction, and on through the oviduct (Birkhead et al. 1993a). Only a minute fraction of the inseminated spermatozoa reach the site where eggs are fertilized, but the actual number varies considerably across species (Birkhead et al. 1994). By counting the number of spermatozoa in or penetrating the perivitelline lav ers of fresh eggs, it is possible to obtain an index of the number of spermatozoa present in the infundibulum at the time of fertilization (Birkhead and Fletcher 1994).

When released from the ovary, the ovum is covered by the inner perivitelline layer. Fertilization takes place 15 to 30 min after ovulation with one of several spermatozoa that penetrate the inner perivitelline layer (Bakst and Howarth 1977). Shortly after fertilization, the outer perivitelline layer is laid down on the ovum, trapping other spermatozoa present in the infundibulum (Wishart 1987). The number of holes in the inner perivitelline layer is correlated with the number of spermatozoa trapped on the outer layer (Birkhead and Fletcher 1994, Birkhead et al. 1994).

In this paper, we report on variation in the number of spermatozoa in eggs of two passerine species, the Blue Tit (Parus caeruleus) and the Great Tit (P major), with a special focus on variation within and between clutches of individual females. The results are discussed in light of the mating system of these species.

Study areas and methods.-Eggs were collected in early May 1997 from two populations of Blue Tits and Great Tits that nested in boxes in southeastern Norway. One study site was located on the island of Jomfruland (58 deg 52'N, 9 deg 36'E) off the coast of Telemark County, where tit populations have been studied for several years (Krokene et al. 1998). On Jomfruland, we collected a maximum of three eggs from each clutch, which did not interrupt the normal nesting events. At the other study site, in Maridalen, Oslo (59 deg 58'N, 10 deg 47'E), we collected entire clutches of both species to study variation in total number of spermatozoa over the laying sequence. For the Blue Tit, the overall sample for analysis consisted of 107 eggs from 21 females (35 eggs from 13 clutches on Jomfruland, and 72 eggs from 8 clutches in Maridalen). For the Great Tit, we analyzed a total of 94 eggs from 26 females (52 eggs from 21 clutches on Jomfruland, and 42 eggs from 5 clutches in Maridalen). We attempted to randomize the eggs collected on Jomfruland with respect to laying order, but because the population was not monitored continuously and eggs often had to be taken before clutches were complete, the eggs collected on Jomfruland were biased toward the earlier part of the laying sequence. For the Blue Tit, eggs analyzed from Jomfruland clutches were on average 1.3 eggs earlier in the laying sequence than those from Maridalen clutches (t - 2.66, df = 19, P = 0.016). For the Great Tit, Jomfruland eggs were on average 1.9 eggs earlier in the laying sequence than those from Maridalen (t = 3.97, df 24, P

Eggs were collected during the laying period, which ensured that they had not been incubated. They were stored at 4 deg C for one to six days until shipped by express courier to TRB's tab in Sheffield for immediate examination. In the lab, eggs were opened with scissors and the perivitelline layers were removed, placed on a microscope slide, and stretched out flat. Next, a drop of the fluorescent Hoechst dve 33342 was added to stain the sperm nuclei, and the layers were examined under a microscope at 20X and 40X power. The number of spermatozoa on the inner and outer perivitelline layer of each egg and the number of holes in the inner perivitelline layer were recorded (see Birkhead et al. 1994). Unfortunately, several eggs were damaged during shipment. Except for two Blue Tit eggs and six Great Tit eggs, however, the perivitelline tissue or a part of it could be rescued and examined. The number of trapped spermatozoa was then extrapolated from counts on the intact part. In a total of 27 Blue Tit eggs and 35 Great Tit eggs, the inner perivitelline layer with the blastodisc could not be inspected. In these cases, we estimated the number of holes from the number of spermatozoa trapped on the outer perivitelline layer, using a regression for intact eggs.

Results.-In both species, there was a positive correlation between the number of holes made by spermatozoa penetrating the inner perivitelline layer and the number of spermatozoa trapped on the outer layer (Blue Tit, r - 0.59, n = 20, P = 0.006; Great Tit, r = 0.64, ti = 24, P = 0.0007). A similar relationship has been documented in other species (Birkhead et al. 1993b, 1994; Birkhead and Fletcher 1994). Holes in the inner perivitelline layer were concentrated on the blastodisc. The proportion of holes located on the blastodisc in eggs with intact membranes amounted to 77% In = 68) in the Blue Tit and 71% (n = 61) in the Great Tit,