Testicular biopsy in psittacine birds (Psittaciformes): impact of endoscopy and biopsy on health, testicular morphology, and sperm parameters.
Key words: testicular biopsy, endoscopy, sperm, avian, cockatiels, parakeets, Psittaciformes, Nymphicus hollandicus, Psittacula krameri
One of the most threatened bird orders worldwide is Psittaciformes, the parrots. (1) Hence, intense research concerning reproductive physiology of these birds is required. Several approaches are available for assessing the reproductive status of male psittacine birds, predominantly sperm analysis (2-11) or endoscopic evaluation of testicular morphology. (12) However, results of part 1 of this study ("Testicular biopsy in psittacine birds [Psittaciformes]: Comparative evaluation of testicular reproductive status by endoscopic, histologic, and cytologic examination") revealed that macroscopic evaluation of the testicles in different psittacine birds is unreliable and that histologic investigation of a testicular biopsy sample is required. (13) Several studies have evaluated testicular histology in different psittacine birds but mainly via intravitam or postmortem removal of testicles. (14-18) The purpose of the present study was to evaluate the viability of testicular sampling from live birds by assessing the impact on the birds' health, testicular integrity, and sperm quality.
Materials and Methods
The study period was November 2006 through December 2007. During this time, 7 male rose-ringed parakeets (Psittacula krameri; 3-7 years old; mean age, 5 years) housed with 3 female parakeets and 9 male cockatiels (Nymphicus hollandicus; 2-5 years old, mean age, 4 years) housed with 2 females were kept in aviaries. Husbandry conditions were given in part 1 of this paper. (13) Briefly, the birds were kept in outdoor aviaries from April to October and in outdoor aviaries during the cold season.
Endoscopy and testicular sampling
A total of 4 endoscopic procedures were performed in each bird at 3-month intervals. Endoscopies were carried out in January, April, July, and October as previously describedy. (13) First, the appearance of the testicular surface and the air sac membranes were evaluated to document pathologic alterations arising from recent endoscopies. A biopsy sample was taken from the cranial part of the left testicle, away from the epididymis, by biopsy forceps measuring 1.7 mm (5 Fr, Karl Storz, Tuttlingen, Germany). When removing the biopsy forceps from the body cavity, counterpressure with the working channel was exerted on the testicle to avoid lacerating the testicular vessels. Any occurrence of apnea, hemorrhage, early termination of endoscopy, intraoperative or postoperative death, or prolonged recovery after anesthesia was documented.
From late February 2007 until December 2007, desemination trials were performed in all males once a week. Semen collection was performed in the morning, according to the established method of cloacal massage in budgerigars (Melopsittacus undulatus). (4,7) Because rose-ringed parakeets and cockatiels are larger and more resolute than budgerigars, an assistant loosely restrained the bird in a half upright position. The cloaca was massaged with the index and middle finger of the right hand, until an active ejaculation occurred or sperm was collected passively due to cloacal pressure. The obtained seminal fluid was collected immediately in a calibrated microcapillary tube (Ringcaps 1-5 [micro]L, Hirschmann Laborgerate GmbH & Co KG, Eberstadt, Germany) held in the left hand of the examiner. Cloacal massage was discontinued in cases in which birds displayed defensive behavior, such as intense struggling or screaming, or if cloacal bleeding occurred. Generally, the massage was stopped after about 10 minutes if sexual excitation of the bird ceased.
The collected semen samples were examined macroscopically and microscopically. Volume, color, consistency, impurities (blood, urine, feces), and pH of the semen samples, as well as motility of spermatozoa, sperm concentration and total sperm count, live-dead ratio, and pathologic changes of spermatozoal morphology, were assessed as described previously in budgerigars. (10)
To evaluate the influence of endoscopy on sperm parameters, the sperm parameters were assessed in intervals of approximately 4 weeks, representing monthly intervals. The postendoscopic interval was only 3 weeks because spermatogenesis in birds lasts up to 3 weeks, including time of passage through the epididymis and ductus deferens. (19,20) Sperm parameters from semen samples obtained within the postendoscopic interval were then compared with those from the samples obtained in the pre-endoscopic interval.
Statistical analysis was conducted by using SigmaPlot 11.0 (Systat Software GmbH, Erkrath, Germany). The Shapiro-Wilk test was used to test normal or non-normal distribution of all data. Significant differences were tested by using the t test (parametrically) or Mann-Whitney U test (nonparametrically). Results were considered significant at P < .05.
Impact of anesthesia and surgery on the health status of the birds
No complications arising from anesthesia, such as apnea, occurred in 64 endoscopic procedures. Among these, 59 surgeries proceeded without complications, and the birds recovered from anesthesia within a few minutes. In the remaining 5 surgical procedures, internal hemorrhage occurred intraoperatively, leading to discontinuation of the endoscopy. Four of these birds recovered quickly and without complications from anesthesia. One rose-ringed parakeet died immediately after endoscopy. Internal bleeding occurred in April, July, and October during culmination in the cockatiels (n = 3) and during quiescence in the rose-ringed parakeets (n = 2).
Impact of testicular biopsy on testicular morphology
No pre-existing defects were seen on the testicles or air sacs during the first endoscopy. Three months later, more than 80% of the cockatiels (8/9) and rose-ringed parakeets (6/7) had no macroscopically visible sequelae resulting from the first endoscopy. Only 1 cockatiel displayed divided testicular tissue (fissured and gaping testicular tissue), and 1 rose-ringed parakeet had testicular cicatrization (scars) at this time. The number of birds with damaged testicles or air sacs, mainly in the form of testicular cicatrization, increased in the subsequent endoscopies (Fig 1). Further macroscopic changes were division of testicular tissue and thickened air sac membranes. In 1 cockatiel, the left testicle was absent at the fourth endoscopy. Above all, 3 of 9 cockatiels and 6 of 7 rose-ringed parakeets showed pathologic findings in the left testicle at the fourth endoscopy. The remaining birds either developed persistent air sac holes or showed no pathologic changes. In 1 case, evaluation was not possible due to early termination of the endoscopy arising from hemorrhage.
Impact of testicular biopsy on sperm production and quality
In the rose-ringed parakeets, semen collection was inconsistent. Only 9 sperm samples were collected from 3 birds. Semen collection was successful in March and in May but completely failed in April. Statistical evaluation of sperm parameters based on the impact of endoscopy was not performed due to the small number of semen samples.
By contrast, semen collection was consistently successful in the cockatiels. The number of semen samples collected each week increased in April and May (Fig 2) because the birds were kept in an outdoor aviary. However, the proportion of passive ejaculations increased predominantly, whereas active ejaculations decreased during the course of the year. Although sperm production of the cockatiels declined in September and October, there was a brief upward trend in November, after the birds were moved back into the indoor aviary. The impact of endoscopy was initially evaluated after the second endoscopy performed in April. This endoscopic procedure had no significant effects on sperm production (Fig 2) and sperm quality (Fig 3). In July, sperm concentration, total sperm count (P < .001), and live-dead ratio of spermatozoa (P < .02) were significantly lower after endoscopy than in the weeks before. Those values did not change in the following months but increased again in early October. Further sperm parameters displayed no alteration after endoscopy. The last endoscopic procedure was performed in late October. At this time, because the testicles were regressing in many cockatiels and sperm quality was low, the procedure had no effect on sperm production and sperm parameters.
A single endoscopic testicular biopsy caused no complications in most of the examined cockatiels and rose-ringed parakeets, analogous to the results in budgerigars, (10) white-eyed parakeets (Aratinga leucophthalma), (21) and larger psittacine birds. (22) Sequelae were rarely observed, and testicular cicatrization and divided testicular tissue occurred most frequently. In subsequent endoscopies, pathologic findings on the left testicle and the air sacs increased. In 1 cockatiel, the left testicle was absent after repeated sampling, indicating loss of testicular tissue via sampling or testicular atrophy. However, this phenomenon would not be expected after a single biopsy. Furthermore, one rose-ringed parakeet died during the fourth endoscopy because of severe bleeding. Nevertheless, bleeding complications occurred to a much lesser extent than in budgerigars (10) and swift parrots (Lathamus discol or), (23) probably because of the body size of the birds. About one-third of the birds showed no macroscopic testicular alterations until the fourth endoscopy.
Analogous to the results obtained in budgerigars, (10) testicular biopsies had only minor effects on sperm parameters. Sperm production stopped transiently around the second endoscopy in the rose-ringed parakeets, and sperm quality decreased after the third endoscopy in the cockatiels. Although testicular biopsy may be causative for these alterations, other factors must be considered. In the rose-ringed parakeets, the period of aspermia in April had begun already before the endoscopic procedure. Again, other factors must be considered, such as stress due to conflicts among the parakeets or the transfer to the outdoor aviaries. In the cockatiels, the affected sperm parameters remained low in the subsequent months but increased again after relocating the birds into the indoor aviaries. Thus, physiologic regression due to climatic changes may also be causative for decreasing sperm quality in July. In budgerigars, only minor effects of testicular biopsy on sperm quality were detected as well. (9) Nevertheless, studies in mammals reveal the occurrence of antisperm antibodies (ASA) after testicular biopsy. (24) The ASA are described to reduce sperm quality in men (25) and in stallions (26) but not in bulls (27) and dogs. (24) Whether ASA occur in psittacines is not known.
Our results show that taking a single biopsy sample from the testicle is a low-risk method for the evaluating the reproductive status of male parrots. Nevertheless, an impact on testicular integrity or on sperm quality cannot completely be ruled out, which is of special significance for endangered species. If taking a testicular biopsy is not required, endoscopic fine-needle aspiration for testicular cytologic examination, which has been described in swift parrots, (23) should be considered as a less invasive alternative.
Acknowledgments: We thank K. Steinbach-Sobiraj and Dr. A. Hahn for their collaboration. This project was supported by the Deutsche Forschungsgemeinschaft, Ei 333/12-2 and Kr 1732/5-1 and licensed by the responsible regional authority (Regierungsprasidium Leipzig, TVV 25/06).
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Maria Hanse, MedVet, Maria-Elisabeth Krautwald-Junghanns, Prof Dr MedVet, Dipl ECZM (Avian), Susanne Reitemeier, MedVet, Almuth Einspanier, Prof Dr MedVet, and Volker Schmidt, Dr MedVet, Dipl ECZM (Avian)
From the Clinic for Birds and Reptiles (Hense, Krautwald-Junghanns, Schmidt) and Institute of Physiological Chemistry (Reitemeier, Einspanier), Veterinary Faculty, University of Leipzig, An den Tierkliniken 17, 04103 Leipzig, Germany.
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|Author:||Hanse, Maria; Krautwald-Junghanns, Maria-Elisabeth; Reitemeier, Susanne; Einspanier, Almuth; Schmidt|
|Publication:||Journal of Avian Medicine and Surgery|
|Date:||Dec 1, 2013|
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