Fertility and Insemination Characteristics of Sperm Storage Tubules in Old Thai-Native Hens

Abstract

We aimed to evaluate the effects of sperm concentration (150-250 × 10⁶ spz/dose) and insemination frequency (once, twice, and thrice weekly) on fertility and sperm storage tubule (SST) characteristics. The SSTs were classified into five categories: namely, SSTs having an unscorable (SST1), empty (SST2), low (SST3), medium (SST4), and high (SST5) sperm count after insemination. The results showed that only insemination frequency affected the fertility rate (p < 0.05). The highest fertility was found in the thrice-weekly insemination group; however, this rate was not significantly different from that for the twice-weekly insemination group, except on day 7, while the once-weekly insemination group showed the lowest fertility rate (p < 0.05) from day four onward. On day 1, the SST characteristics showed no differences among the various insemination frequencies. On day 4, the SST2 and SST3 categories increased in the once-weekly insemination group (p < 0.05), while the SST4 and SST5 categories decreased compared to the twice- and thrice-weekly insemination groups (p < 0.05). On day 7, only the thrice-weekly insemination group maintained a level of SST5 category tubules like that measured on day 1 (p > 0.05). In summary, the insemination dose of 150 × 10⁶ sperm was enough for fertilization, and thrice-weekly insemination was the appropriate frequency in old Thai native hens for maintaining a high sperm density in the SSTs throughout the week.

Keywords: Thai indigenous chicken; fertility rate; insemination frequency; semen concentration.

Figure 1

Morphology of Pradu Hang Dum chickens: male (left side) and female (right side). Overall, the ideal traits are strong, with the feathers covering most of the body being black, with red-to-dark brown feathers on top, mostly black eyes, black ear lobes, yellow and black beaks, red face and pea comb, and black shanks. The dark brown-to-black toes and claws should ideally be black.

Figure 2

Experimental design and sample collection. The optimal sperm concentration dosage and insemination frequencies were examined by a fertility test (a). After finishing the fertility test, the resident sperm in the UVJ after insemination was examined by means of the sperm-containing SST test (b).

Figure 3

Transverse section of the uterovaginal junction (UVJ) fold of an old Thai native hen after insemination for at least 24 h containing sperm storage tubules (SSTs; black arrowhead) stained with hematoxylin and eosin (H & E) (A). Classification of SSTs within the UVJ section for sperm storage was assessed under a digital pathology scanner with computer-assisted software for image analysis. The samples were categorized into five groups: SST1 consisting of SSTs that could not be identified (unscorable; (a)), SST2 without sperm (empty; (b)), SST3 with 1–5 sperm cells (low; (c)), SST4 containing 6–20 sperm cells (medium; (d)), and SST5 with more than 20 sperm cells (high; (e)), respectively. S, sperm. Scale bars = 100 µm (A) and 20 µm (a–e). BV—blood vessel; E—epithelium; L—lumen, LP—lamina propria; and S—stoma.

Figure 4

Fertility percentage (%) ± SEM comparison among different insemination frequencies (once, twice, and thrice weekly). ^a,b,c Different letters within a day after artificial insemination indicate significant differences (p < 0.05). The values are percentages of fertilized eggs per group and collection day. Freq1 = once-weekly insemination; Freq2 = twice-weekly insemination; and Freq3 = thrice -weekly insemination.

Figure 5

Effect of insemination frequency on SST sperm content characteristics in UVJ tissue after insemination at day 1, day 4, and day 7. ^a,b,c,d different letters indicate significant differences within each SST category (p < 0.05). (a) SST1 = SSTs containing sperm that could not be identified; (b) SST2 = SSTs without sperm; (c) SST3; SSTs with 1–5 sperm cells; (d) SST4; SSTs with 6–20 sperm cells; (e) SST5; SSTs with more than 20 sperm cells. Freq1; once-weekly insemination; Freq2; twice-weekly insemination; and Freq3; thrice-weekly insemination.