Fig 1.
Semen collection in kākāpō using abdominal massage technique from two different points of view.
A male kākāpō is manually restraint, carefully wrapped in a towel and with the head and chest inserted in a bottom removed water bottle.
Fig 2.
Semen collection from the cloaca of a kākāpō using pipettes and plastic pipette tips.
Using thumb and index finger the cloaca is gently pressed to carefully squeeze the semen out of the cloaca. Assistants help to hold feathers away in order to prevent that semen is soaked up by the feathers.
Fig 3.
Semen collection in kākāpōs using electric stimulation.
A bodysize-adapted multipolar probe was inserted into the urodeum of the cloaca in order to stimulate the ampullae ductus deferentes and to collect semen using a slight electrical current.
Fig 4.
Semen analysis was performed immediately in the field using mobile equipment. This included a microscope with 40 X - 400 X magnification and phase contrast and a heating pad attached to a 12V mobile motorcycle battery. The individuals depicted in this manuscript have given written informed consent (as outlined in PLOS consent form) to publish this picture and these case details.
Fig 5.
Artificial insemination in a kākāpō.
A) Birds were restrained in dorsal recumbency with their head and chest elevated and their feet gently pulled towards their head. Feathers are taped out of the way using a weakly adhesive tape. B) Veterinary elastrator pliers were used for insertion into the cloaca, spreading the cloaca for visualization of the oviductal opening to enable semen deposition. C) Inside the spread cloaca the oviductal opening (left body side) must be recognized and differentiated from the intestinal opening (left body side), being slightly darker and may show feces in the lumen or directly in front as on this picture.
Table 1.
Mean body weight, age, handling times and semen parameters of male Kākāpō (Strigops habroptilus) (n = 20).
Table 2.
Comparative semen evaluation of male Kākāpō (Strigops habroptilus) collected through two different methods of semen collection.
Table 3.
Evaluation of individual semen parameters of repeatedly captured Kākāpō (Strigops habroptilus) males (n = 11), in ascending order of their genetic priority.
Table 4.
Statistical evaluation of the number of morphological semen characteristics per slide of male Kākāpō (Strigops habroptilus). In each slide (N = 33) 200 spermatozoa were evaluated.
Table 5.
Reevaluation of MOT, PMOT and viability using different semen extenders in Kākāpō (Strigops habroptilus) semen samples under the aspect of storage time (in hours (h).
Fig 6.
Assessment of sperm motility in diluted semen samples from kākāpō comparing fresh semen (Time point 0) to stored-semen after 24, 48, 72, and 96 hours storage time at + 4 °C in a refrigerator.
As diluents isotonic saline (NaCl) 1), Glutac-2 (Glutac), modified Blanco`s semen extender (Blanc), 1% Glucose-Ringer`s® solution (Glucose 1%), modified Beltsville poultry semen extender (BPSE) and modified Lake semen extender (Lake) were used. Motility (MOT) depicts the percentage of all motile spermatozoa compared to the total number of spermatozoa, whereby the percentage of progressively forward moving Spermatozoa from all spermatozoa is depicted in the progressive forward motility graph (PMOT).
Fig 7.
Assessment of sperm viability using Eosin B stain in diluted semen samples from kākāpō, comparing a fresh semen (Time point 0) to stored semen after 24, 48, 72, and 96 hours storage time at + 4 °C in a refrigerator.
As diluents isotonic saline (NaCl) 1), Glutac-2 (Glutac), modified Blanco`s semen extender (Blanc), 1% Glucose-Ringer`s® solution (Glucose 1%), modified Beltsville poultry semen extender (BPSE) and modified Lake semen extender (Lake) were used. The percentage of viable spermatozoa is depicted compared to the total number of spermatozoa.
Table 6.
Mean body weight, age and handling times of female Kākāpō (Strigops habroptilus) (n = 12), as well as standard semen parameter of semen samples used for artificial insemination.
Table 7.
Artificial inseminations (n = 15) performed in Kākāpō (Strigops habroptilus) females (n = 12), including semen quality parameter of semen samples and semen extenders used, as well as egg fertilization successes through AI (proven through paternity testing).
Table 8.
Clutch and egg fertility rates in two groups (AI, n = 5; Control, n = 7) of female Kākāpō (Strigops habroptilus) differentiated between first and second clutch.