Raptors bred in captivity: semen characteristics and assisted reproduction outcome in goshawk (Accipiter gentilis)

Abstract

Three sexually mature goshawks reared in captivity and imprinted on humans to express reproductive behavior according to the cooperative method were studied for three consecutive breeding seasons to assess the quality of their sperm. The following parameters were analyzed: ejaculate volume and sperm concentration, motility, viability, and morphology. Ejaculate volume, sperm concentration and motility fluctuated along the reproductive season, revealing the greatest quality of the reproductive material at full springtime (i.e., April). Motility of the sperm collected in March strongly reduced with age, contrary to samples collected in April or May. Sperm viability was not influenced by either age or month of collection within each season. Ultrastructural investigations provided information on normal sperm morphology for the first time in this species. The morphological categories of sperm defects in fresh semen, present at low percentages, are also described. Functional analyses (perivitelline membrane assay and artificial inseminations) confirmed the good quality of the semen obtained using the cooperative method. The reported data provide the basis for further studies aimed at developing protocols to improve the outcome of artificial insemination and semen cryopreservation in the goshawk as well as other bird of prey species.

Keywords: Captive breeding; Goshawk; Raptor species conservation; Season; Semen characteristics; Sperm ultrastructure.

Figure 1. Effect of goshawk age (A, in years) and reproductive season month (M) on ejaculate volumes (LSmean ± Standard Error Estimates of the LSmeans (SEE)).

LSmeans identified by identical letters were not considered significantly different according to Tukey-Kramer conservative (when applicable) grouping at the 5%. The grouping procedure did not reflect all significant comparisons. The following additional pairs appeared significantly different according to the p-difference: M3A6 vs. M5A6. See Supplemental Materials for the raw results of the statistical analysis and the arithmetic means and computed standard errors.

Figure 2. Effect of goshawk age (A, in years) and reproductive season month (M) on sperm concentrations (LSmean ± Standard Error Estimates of the LSmeans (SEE)).

LSmeans identified by identical letters were not considered significantly different according to Tukey-Kramer conservative (when applicable) grouping at the 5%.The grouping procedure did not reflect all significant comparisons. The following additional pairs appeared significantly different according to the p-difference: M4A7 vs. M3A8; M3A9 vs. M5A6; M3A8 vs. M5A6; M5A8 vs. M5A6; M3A6 vs. M5A6; M3A7 vs. M5A6. See Supplemental Materials for the raw results of the statistical analysis and the arithmetic means and computed standard errors.

Figure 3. Effect of goshawk age (A, in years) and reproductive season month (M) on sperm motility (LSmean ± Standard Error Estimates of the LSmeans (SEE)).

LSmeans with a letter in common should not be considered different according to Tukey-Kramer conservative (when applicable) grouping at the 5%. The grouping procedure does not reflect all significant comparisons. See Supplemental Materials for the raw results of the statistical analysis and the arithmetic means and computed standard errors.

Figure 4. Effect of goshawk age (A, in years) and reproductive season month (M) on sperm viability (LSmean ± Standard Error Estimates of the LSmeans (SEE)).

Tukey-Kramer conservative (when applicable) grouping at the 5% did not retrieve differences among treatments despite the M(A) interaction term showed a p = 0.0002. Unadjusted p-differences can be found in the Supplemental Material table “SAS results” from line 1,028 to line 1,135. See Supplemental Material for the raw results of the statistical analysis and the arithmetic means and computed standard errors.

Figure 5. In vitro goshawk sperm penetration ability.

In vitro goshawk sperm penetration ability assessed using the sperm-egg interaction assay on IPL from chicken eggs. The test was performed only on the last year of essay for each individual (Gos-7 = 7-year-old goshawk, Gos-8 = 8-year-old goshawk, Gos-9 = 9-year-old goshawk).

Figure 6. Electron microscopy characterization of the spermatozoon of A. gentilis.

SEM micrographs (A) of the entire filiform sperm cell, and (B) the head with the acrosome at the anterior tip. (C) Longitudinal sections of the sperm head by TEM. (D–G) Transversal sections at different levels of the sperm head. (H) SEM image of the transition region from the midpiece and the principal piece of the tail. TEM micrographs of respectively the midpiece in longitudinal (I) and cross sections (J–L), and of the principal piece in longitudinal (N) and cross sections (M, O and P). The principal piece of the tail finishes in a short and thinner endpiece (P). Acrosome (a), annulus (arrows), axoneme (ax), dense fibers (df), distal centriole (dc), fibrous sheath (fs), midpiece (MP), mitochondria (m), nucleus (n), perforatorium (p), principal piece (PP), proximal centriole (pc), and rostrum (r). (A) bar: 10 μm. (B) bar: 1 μm. (C–G) bars: 250 nm. (H) bar: 1 μm. (I–L) bars: 250 nm. (M) bar: 100 nm. (N) bar: 250 nm. (O and P) bars: 100 nm.

Figure 7. Electron microscopy images of sperm head defects.

SEM and TEM images (A and B) of cytoplasmic droplets associated with a weak head bending. (C and D) Morphology and ultrastructure of a severe bending of the head at the proximal region of the midpiece. (E–H) Spermatozoa characterization by SEM and TEM of round heads with a spherical shape. (I and J) Sperm abnormalities on the acrosome shown by SEM, indicating the complete absence of and a swollen acrosomal cap, respectively. (K) TEM image of a nuclear defect, with indentations of the chromatin and the absence of the plasma membrane. Cytoplasmic droplet (*), nucleus (n). (A) bar: 1 µm. (B) bar: 250 nm. (C) bar: 1 µm. (D) bar: 250 nm. (E) bar: 1 µm. (F) bar: 250 nm. (G) bar: 1 µm. (H) bar: 250 nm. (I) bar: 1 µm. (J) bar: 1 µm. (K) bar: 250 nm.

Figure 8. Electron microscopy images of sperm tail defects.

SEM and TEM micrographs (A and B) of abnormalities showing the midpiece with a breakage or the absence of the plasma membrane which revealed the individual mitochondria. (C) Abnormalities involving the principal piece of the tail with coiling. (D) Tail forming a loop at the point of folding. (E–G) SEM and TEM images of a tail wrapping around the head, and the plasma membrane covering both structures, as evidenced in cross sections at various levels of the sperm. Acrosome (a), axoneme (ax), mitochondria (m) and nucleus (n). (A) bar: 1 μm. (B) bar: 250 nm. (C–E) bars: 1 μm. (F and G) bars: 250 nm.