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Semen collection and spermatozoa characteristics in the kea parrot (Nestor notabilis).

Abstract: We describe the seminal characteristics of the kea parrot (Nestor notabilis), an endangered species endemic to the South Island of New Zealand. Semen was collected in the full breeding season from 6 birds in the collection of an amateur aviculturist. The manual massage technique was used. A total of 25 ejaculates was collected and evaluated for volume, degree of contamination, and spermatozoa concentration; motility and kinetic parameters were assessed on diluted samples (modified Tyrode's Albumin Lactate Pyruvate, pH 8.2, temperature 37.5[degrees]C) with a computer-aided sperm analyzer. Four ejaculates were not analyzed because of an excessively high degree of contamination. Semen color ranged from transparent or turbid yellow to whitish. The geometric mean of spermatozoa number/ejaculate was 765.9 [+ or -] 2084.7 X [10.sup.6]. Total and progressive motility were 71.7% [+ or -] 20.0% and 59.8% [+ or -] 22.0%, respectively. Great variability was observed both among birds and among different ejaculates of the same subject. The seminal characteristics of kea are worth further investigation, with the aim of relating semen quality to fertility and defining a minimum inseminating dose for breeding purposes. A deeper knowledge of male reproductive biology also can increase the success of breeding programs and enable the use of the kea as a model species for other more threatened species, such as the kaka (Nestor meridionalis) and the kakapo (Strigops habroptila).

Key words: avian semen, computer-assisted semen analysis, avian, kea parrot, Nestor notabilis

Introduction

The kea (Nestor notabilis) is a large endangered parrot, endemic to the South Island of New Zealand. It lives in high-altitude forest and alpine basins ranging between 600 and 3000 m above sea level and is the only psittacine bird living in an alpine environment. (1) The kea belongs to the subfamily Nestorinae, which is endemic to New Zealand and contains only one other species, the kaka (Nestor meridionalis). The kea is the largest flighted terrestrial bird in New Zealand and the second largest parrot in the country after the kakapo (Strigops habroptila). (1) The kea gained full protection status in 1986 under the Wildlife Act of 19532and is now listed as a Nationally Endangered species by the Department of Conservation under the New Zealand Threat Classification List. (3) It is listed as Vulnerable by the International Union for Conservation of Nature Red List. (4) The current population is estimated to be approximately 10005000 birds. (5) The population of kea has been strongly influenced by human activities and, because of frequent attacks on sheep, more than 150 000 birds were killed as a result of a governmental bounty scheme between 1890 and 1970. (6) This parrot is not currently threatened, although the species is known to be in decline because of degradation of its habitat through fire and overgrazing, predation on chicks by newly introduced mammals such as stoats, illegal shooting and poisoning to protect human activities, and illegal capture and trading. (7) To date, no recovery program has been implemented for the kea.6 However, monitoring is important to continue so that any significant decline can be promptly detected and appropriate conservation actions taken. A thorough knowledge of the reproductive aspects of any species is crucial to carry out captive breeding programs, and artificial insemination is considered an effective tool for ex situ conservation of endangered avian species. (8) Semen analysis and characterization are the prerequisite to semen handling and artificial insemination. The kea can also represent an interesting study model for similar threatened species, such as the kaka and the kakapo. The aim of this study was to characterize the semen of keas kept in captivity. The method used for the work was computer-aided sperm analyzer (CASA) analysis.

Materials and Methods

The kea included in this study were in the collection of an amateur aviculturist. All the birds had been tested with different polymerase chain reaction protocols and were found to be free from avian polyomavirus, (9) psittacine beak and feather disease (circovirus), (10) and psittacosis. (11) All birds were clinically healthy, and their body condition was good. Six males aged 2 to 5 years were evaluated through celioscopy to exclude any gonadal pathology and to assess gonadal maturity through the observation of size, color, and degree of vascularization of both testes and degree of development of both epididymes and vas deferens. Three birds (birds 1, 2, 3) were housed in a large aviary (10 [m.sup.2]) with a young, sexually mature female, while each of the other 3 males (birds 4, 5, 6) were housed as a pair with a female in a 6 X 2 X 2-m aviary. The parrots were fed with the same commercial extruded diet (All Pet, Caronno Pertusella, Varese, Italy) in a ration equal to 20% of their body weight, enriched with boiled rice salad with different fresh fruits and vegetables (apple, grapes, carrot, zucchini, corn).

Semen was collected in the full breeding season (January to May) over a period of 12 weeks, with a weekly collection attempt for each parrot, for a total number of 72 collection attempts. For semen collection, each bird was physically restrained by an operator by means of a soft towel to restrain the head and neck of the bird, to avoid struggling and to work safely. Ejaculation was obtained by massaging, with the thumb and index or middle finger, the rump sliding toward the abdomen near the pubic bones, followed by gentle rhythmic squeezing at the base of the cloaca with the same finger of the other hand. (12,13) The ejaculate was collected in graduated microcapillary tubes (Microcaps, Drummond Scientific Co, Broomall, PA, USA) and semen volume was recorded. Immediately after collection, semen was empirically diluted depending on the expected spermatozoa concentration after preliminary trials (data not shown). The semen was diluted 1:10 with modified TALP (100 mM sodium chloride, 3.1 mM potassium chloride, 25 mM sodium carbonate, 0.3 mM sodium dihydrogen phosphate, 10 mM Hepes, 2 mM calcium chloride, 0.4 mM magnesium chloride and 1 mg/mL sodium pyruvate; Sigma-Aldrich, St Louis, MO, USA), calibrated to pH 8.2, and maintained at 37.5[degrees]C. The time from semen collection to analysis was within 5 minutes. The degree of contamination of the diluted ejaculates was visually classified from 1 to 5, and the type of contaminant was recorded (urates and/or erythrocytes). Ejaculates showing grade 4 or 5 contamination were discarded. Sperm concentration was determined with a Makler chamber after 1:50 or 1:100 dilution of 10 [micro]L of the extended sample with a solution of distilled water and 4% formaldehyde. Semen motility and motility characteristics were evaluated by a CASA (CEROS, Hamilton Thorne Research Inc, version 14, Build 008, IMV Technologies, L'Aigle, France) on 10 [micro]L of extended semen placed in a preheated Makler chamber (37.5[degrees]C). The parameters evaluated were total motility (TM, %), progressive motility (PM, %), average path velocity ([micro]m/s), straight-line velocity ([micro]m /s), curvilinear line velocity (([micro]m/s), amplitude of lateral head displacement ([micro]m), beat cross frequency (Hz), straightness of track (%), and linearity of track (%). The settings of the instrument were as follows: 60 frames per second (Hz), 30 frames per field, minimum contrast 1/4 20, minimum cell size 1/48. Static cells were defined as having an average path velocity less than 5 [micro]m/s and straight-line velocity less than 13 [micro]m/s. These parameters were chosen after different trials with kea semen (data not shown). All the biological material used to perform the present study was collected for diagnostic purposes by standard clinical procedures. The study was performed in accordance with the guidelines for the care and use of animals of the Department of Veterinary Science of the University of Turin, Italy. Previous informed consent was obtained from the owners.

Results

A different number of examinable ejaculates could be collected from the 6 males, and the 3 keas that were housed in pairs with the females gave a greater number of samples. A total of 25 ejaculates was collected, but four samples were not analyzed because of a contamination degree higher than 4 (Table 1). The collection success rate ranged from a minimum of 8.3% (kea no. 2) to a maximum of 58.3% (kea no. 6). Semen color ranged from transparent or turbid yellow to whitish, depending on sperm concentration. Table 2 shows the mean values ([+ or -] SD) of volume, degree of contamination, and spermatozoa number in the ejaculates of each of the 6 birds and the geometric mean of all the ejaculates. Contamination occurred in all samples and was mainly due to erythrocytes and urates; it was evaluated as grade 1 in 32% of the samples, grade 2 in 40%, grade 3 in 12%; grade 4 or 5 in 16%. The presence of blood in the ejaculates was mainly due to the fragility of the cloacal mucosa capillaries but had no consequences for the health of the birds.

The geometric mean of the spermatozoa number was 765.9 [+ or -] 2084.7 x [10.sup.6] sperm; higher values were measured in kea 6 (996.4 [+ or -] 762.5 x [10.sup.6] sperm) and lower values in kea 1 (115 X [10.sup.6] sperm). The numerically highest sperm concentration was measured in kea 3, a value of 550 X [10.sup.6] sperm/mL, while the lowest was in kea 4, with 10 x [10.sup.6] sperm/mL. Total and progressive spermatozoa motility, together with the kinetic parameters, are listed in Table 3. The highest mean values for TM belonged to kea 6 (TM: 82.0% [+ or -] 18.4%; PM: 70.0% [+ or -] 17.4%) and the lowest to kea 2 (39.0%) for TM and to kea 3 for PM (22.0%). However, these observations are limited to the single sample that could be analyzed.

Discussion

Although the kea is one of the most studied psittacine species and among the most desired by any collector and parrot breeder, its seminal characteristics have never been published. A deeper knowledge of male reproductive biology can increase the success of breeding programs and enable the use of the kea as a model species for other more threatened species, such as the kaka and kakapo. In our work, we evaluated kea semen by a standardized and objective method, the CASA system.

In the absence of previous studies of this subject in the kea, we can compare our results with those reported for other analogous species. The success rates of the attempts at semen collection appear to vary among species, namely, 74.2% in cockatiels (Nymphicus hollandicus), (14) from 34.9% to 83.3% in Hispaniolan Amazon parrots (Amazona ventralis), (15) and from 54.5% to 96.6% in other species. (16) In our case, the best donor had a success rate of approximately 60%. Semen collection success is also influenced by the method of collection. The collection technique that we adopted is similar to that reported by Samour (17) except for our use of a softer device and the aid of a second operator. In accordance with the guidelines for the care and use of animals of the Department of Veterinary Science of the University of Turin, we did not consider electro-ejaculation, although it has been previously used in psittacine birds. (16,18) The degree of ejaculate contamination was seldom high enough to hamper semen analysis in our work. Data in the literature regarding percentages of contaminated ejaculates are limited (9.9% in cockatiels) (14); more often, previous studies only state that contaminated samples were excluded from examination.

The sperm concentration that we measured for kea is higher than the values previously reported in monk parakeets (Myiopsitta monachus), (19) Hispaniolan Amazon parrots, (15) blue-fronted Amazon parrots (Amazona aestiva), (20) and golden-capped conures (Aratinga auricapillus) (21) but much lower than the reported values for cockatiels, (22) budgerigars (Melopsittacus undulatus), (23) and peach-faced lovebirds (Agapornis roseicollis). (13)

The use of a CASA system for the objective evaluation of motility and kinetic parameters of small psittacine bird semen is limited to studies involving budgerigars, (23) cockatiels, (22) and peach-faced lovebirds. (13) In a recent study in budgerigars, Gloria et al (23) showed different semen volumes and motility parameters depending on the husbandry method, with higher values when parrots were kept in pairs with a single female instead of in promiscuous aviaries.

In our study, we made similar observations beginning from the collection success rate; because the birds kept in promiscuity gave a very limited number of samples, volume and motility data were limited to the single samples that could be analyzed from each of the three birds, so no comparison was possible. Flocking type, either in pairs or in groups, can affect semen quality, but several other factors can have an effect on the characteristics of semen samples, such as previous matings, previous semen collections, and season, as reported both in similar species (13) and in other phylogenetically close avian orders. (24)

The motility and kinetic parameters that we measured for kea are numerically higher than the values reported for cockatiels22 but lower than those of budgerigars and peach-faced lovebirds. (13,23) Other reported data regarding sperm motility in Psittaciformes are limited to a few species, and the values, measured by direct microscopic observation, are quite variable: His paniolan Amazon parrots (33%-82%), (15) goldencapped conures (39%-54%), and blue-naped parrots (Tanygnathus lucionensis) (83%-88%). (21)

We observed great variability both among individual birds, some of which could be selected as potential semen donors, and among different ejaculates from the same subject. This individual variability could be caused by several factors (eg, mating before collection). The seminal characteristics of kea merit further investigation with the aim of relating semen quality to fertility and defining a minimum inseminating dose both for breeding purposes and for research regarding the challenges of assisted reproduction in captivity.

Andrea Dogliero, DVM, Ada Rota, DVM, PhD, Dipl ECAR, Renato Lofiego, DVM, Mitzy Mauthe von Degerfeld, DVM, PhD, and Giuseppe Quaranta, DVM, PhD

From the Department of Veterinary Science, University of Turin, Largo Paolo Braccini 2-5, 10095 Grugliasco (TO), Italy.

Acknowledgments: We thank the breeder, Ugo Brambilla, for his invaluable help and cooperation. Thanks to aviculturists like him, a species that is so coveted and yet so rare was raised and maintained with great care, allowing a better understanding of its reproductive biology in captivity.

References

(1.) Forshaw JM. Parrots of the World: An Identification Guide. Princeton, NJ: Princeton University Press; 2006.

(2.) Akers K, Orr-Walker T. The kea. New Zealand's endangered alpine parrot. Kea Conserv Trust. 2009; 9:1-13.

(3.) Hitchmough R, Bull L, Cromarty P. New Zealand Threat Classification Lists--2005. Wellington, New Zealand: Department of Conservation; 2007.

(4.) Bird Life International, 2006. Nestor notabilis. In: 2007 International Union for Conservation of Nature (IUCN) Web site. The IUCN Red List of Threatened Species, www.iucnredlist.org. Accessed June 20, 2015.

(5.) Anderson R. Keas for keeps. For Bird (Wellington). 1986; 17(1):2-5.

(6.) McLelland JM, Reid C, Mclnnes K, et al. Evidence of lead exposure in a free-ranging population of kea (.Nestor notabilis). J Wildl Dis. 2010;46(2):532-540.

(7.) Dussex N, Wegmann D, Robertson BC. Postglacial expansion and not human influence best explains the population structure in the endangered kea (.Nestor notabilis). Mol Ecol. 2014;23(9):2193-2209.

(8.) Blanco JM. Wildt DE, Hofle U, et al. Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species. Theriogenology. 2009;71(1):200-213.

(9.) Phalen DN, Wilson VG, Graham DL. Polymerase chain reaction assay for avian polyomavirus. J Clin Microbiol. 1991;29(5): 1030-1037.

(10.) Ypelaar I, Bassami MR, Wilcox GE, Raidal SR. A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus. Vet Microbiol. 1999;68(1-2): 141-148.

(11.) Chahota R, Ogawa H, Mitsuhashi Y, et al. Genetic diversity and epizootiology of Chlamydophila psittaci prevalent among the captive and feral avian species based on VD2 region of ompA gene. Microbiol Immunol. 2006;50(9):663-678.

(12.) Burrows WH, Quinn JP. A method of obtaining spermatozoa from the domestic fowl. Poult Sci. 1935;14(4):251-253.

(13.) Dogliero A. Use of computer-assisted semen analysis for evaluation of rosy-faced lovebird (Agapornis roseicoliis) semen collected in different periods of the year. Theriogenology. 2015;83(1): 103 106.

(14.) Neumann D, Kaleta EF, Lierz M. Semen collection and artificial insemination in cockatiels (Nymphicus hollandicus)--a potential model for psittacines. Tierarztl Prax Ausg K Kleintiere Heimtiere 2013; 41 (2): 101-105.

(15.) Brock M. Semen collection and artificial insemination in the Hispaniolan parrot (Amazona ventralis). J Zoo Wildl Med. 1991;22(1): 107-114.

(16.) Lierz M, Reinschmidt M, Muller H, et al. A novel method for semen collection and artificial insemination in large parrots (Psittaciformes). Sci Rep. 2013;3:2066.

(17.) Samour JH. The reproductive biology of the budgerigar (Melopsittacus undulatus): semen preservation techniques and artificial insemination procedures. J Avian Med Surg. 2002; 16(1):39-49.

(18.) Harrison GJ, Wasmut D. Preliminary studies of electrostimulation to facilitate manual semen collection in psittacines. Proc Annu Conf Assoc Avian Vet. 1983:207-213.

(19.) Anderson SJ, Bird DM, Hagen MD. Semen characteristics of the Quaker parakeet (Myiopsitta monachus). Zoo Biol. 2002;21(5):507-512.

(20.) DellaVolpe A, Volker S, Krautwald-Junghanns ME. Attempted semen collection using the massage technique in blue-fronted Amazon parrots (Amazona aestiva aestiva). J Avian Med Surg. 2011;25(1): 1-7.

(21.) Stelzer G, Crosta L, Biirkle M, Krautwald-Junghanns ME. Attempted semen collection using the massage technique and semen analysis in various psittacine species. J Avian Med Surg. 2005;19(1):7-13.

(22.) Fischer D, Neumann D. Wehrend A, Lierz M. Comparison of conventional and computer-assisted semen analysis in cockatiels (Nymphicus hollandicus) and evaluation of different insemination dosages for artificial insemination. Theriogenology. 2014;82(4): 613-620.

(23.) Gloria A, Contri A, Carluccio A, et al. The breeding management affects fresh and cryopreserved semen characteristics in Melopsittacus undulatus. Anim Reprod Sci. 2014; 144(1-2):48-53.

(24.) Cheng FP, Guo TJ, Wu JT, et al. Annual variation in semen characteristics of pigeons (Columba livia). Poult Sci. 2002;81(7): 1050-1056.
Table 1. Individual success rate of semen collection
in six kea in 12 semen collection attempts.

            Semen      Individual   Analyzable
          collection    success       semen
Kea no.   success, n    rate (%)     samples

1             2           16.6          1
2             1           8.3           1
3             4           33.3          1
4             5           41.6          5
5             6           50.0          6
6             7           58.3          7

Table 2. Semen volume, degree of contamination, and
spermatozoa number (mean [+ or -] SD) in the ejaculates
(n) of 6 kea (birds no. 1-6).

                                 Kea no.

                          1         2         3
Parameter              (n = 1)   (n = 1)   (n = 1)

Volume, [micro]L         2.3      10.0       9.2

Contamination             2         1         1
degree, l-5 (a)

Spermatozoa number/      115      5478       461
ejaculate, x
[10.sup.6]

                                         Kea no.

                                4                      5
Parameter                    (n = 5)                (n = 6)

Volume, [micro]L         7.9 [+ or -] 8.3       5.1 [+ or -] 3.0

Contamination            1.4 [+ or -] 0.5       1.8 [+ or -] 0.8
degree, l-5 (a)

Spermatozoa number/    321.0 [+ or -] 260.7   241.5 [+ or -] 144.3
ejaculate, x
[10.sup.6]

                             Kea no.

                                6               Geometric mean of
Parameter                    (n = 7)               the 6 birds

Volume, [micro]L        20.6 [+ or -] 31.4      11.2 [+ or -] 6.3

Contamination            2.1 [+ or -] 0.7       1.7 [+ or -] 0.5
degree, l-5 (a)

Spermatozoa number/    996.4 [+ or -] 762.5   765.9 [+ or -] 2084.7
ejaculate, x
[10.sup.6]

(a) Degree of contamination visually classified
from 1 to 5, and the type of contaminant was recorded
(urates and/or erythrocytes). Ejaculates showing grade 4 or
5 contamination were discarded.

Table 3. Semen motility and kinetic parameters
(mean [+ or -] SD) measured by the CEROS analyzer:
values obtained in the ejaculates (n) of 6 kea (birds no. 1-6).

                                Kea no.

Parameter          1 (n = 1)   2 (n = 1)   3 (n = 1)

TM, %                40.0        39.0        78.0
PM, %                25.0        34.0        22.0
VAP, [micro]m/s       --         40.8        78.6
VSL, [micro]m/s       --         32.3        43.3
VCL, [micro]m/s       --         62.8        121.5
ALH, [micro]m         --          3.2         6.8
BCF, Hz               --         19.2        13.9
STR, %                --         78.0        66.0
LIN, %                --         52.0        46.0

                                   Kea no.

Parameter              4 (n = 5)            5 (n = 6)

TM, %              80.6 [+ or -] 7.7    62.1 [+ or -] 22.2
PM, %              70.6 [+ or -] 11.1   55.3 [+ or -] 16.0
VAP, [micro]m/s    56.5 [+ or -] 8.5    49.7 [+ or -] 6.1
VSL, [micro]m/s    48.7 [+ or -] 9.8    43.0 [+ or -] 7.8
VCL, [micro]m/s    78.6 [+ or -] 7.6    75.2 [+ or -] 7.1
ALH, [micro]m       3.7 [+ or -] 0.2     3.6 [+ or -] 0.5
BCF, Hz            22.7 [+ or -] 1.4    23.5 [+ or -] 2.5
STR, %             83.0 [+ or -] 5.2    85.3 [+ or -] 5.5
LIN, %             64.4 [+ or -] 13.6   59.0 [+ or -] 8.3

                        Kea no.

Parameter              6 (n = 7)          Geometric mean

TM, %              82.0 [+ or -] 18.4   71.7 [+ or -] 20.0
PM, %              70.0 [+ or -] 17.4   59.8 [+ or -] 22.0
VAP, [micro]m/s    65.1 [+ or -] 25.7   57.8 [+ or -] 14.5
VSL, [micro]m/s    57.5 [+ or -] 26.2   49.0 [+ or -] 9.2
VCL, [micro]m/s    88.9 [+ or -] 24.1   82.5 [+ or -] 22.2
ALH, [micro]m       4.1 [+ or -] 0.7     3.9 [+ or -] 1.4
BCF, Hz            27.9 [+ or -] 5.0    24.2 [+ or -] 5.2
STR, %             83.9 [+ or -] 6.8    82.9 [+ or -] 7.9
LIN, %             61.7 [+ or -] 12.0   60.3 [+ or -] 7.5

Abbreviations: TM indicates total motility; PM, progressive
motility; VAP, velocity average pathway; VSL, straight-line
velocity; VCL, curvilinear velocity; ALH, amplitude lateral
head; BCF, beat cross frequency; STR, straightness;
LIN, linearity.
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Author:Dogliero, Andrea; Rota, Ada; Lofiego, Renato; von Degerfeld, Mitzy Mauthe; Quaranta, Giuseppe
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:8NEWZ
Date:Mar 1, 2017
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