Susan Clubb, DVM, Dipl ABVP (Avian), Julia Zaias, DVM, Carolyn Cray, PHD, Leonard Berube, and Lorenzo Crosta, DVM
Session #4020
Summary Style Manuscript
Affiliation: From the Rainforest Clinic for Birds & Exotics, PO Box 508 Loxahatchee, FL 33470, USA (Clubb), University of Miami, Division of Comparative Pathology, PO Box 016960 (R-46), Miami, FL 33101, USA (Zaias, Cray), Kissimmee Diagnostic Laboratory, Florida Dept of Agriculture, PO Box 458006, Kissimmee, FL 34745, USA (Berube), and Loro Parque, Loro Parque Blvd, Puerto de la Cruz, Tenerife, Canary Islands, Spain (Crosta).
Introduction
Infertility in psittacines, as indicated by infertile eggs, is a common problem in aviculture. Clinically practical methods for evaluation of fertility in male psittacines have not been reported. In this study, pairs of psittacines were selected for evaluation that had a history of 2 or more years of infertile egg production in an appropriate avicultural setting. While the focus was on male infertility, in most cases the females were examined by endoscopy at the same time as their mates. In a few cases, the ovaries were also biopsied.
The species evaluated were predominately Amazon parrots (Amazona species); however, macaws (Ara species) and Eclectus parrots (Eclectus roratus) were included.
All birds were evaluated prior to biopsy by routine physical and endoscopic examination. Birds selected for the study were anesthetized with isoflurane for endoscopy. Testes were biopsied using the Storz endoscopic system (Karl Storz Veterinary Endoscopy, Inc, Goleta, CA, USA) with biopsy sleeve and 3-mm cup biopsy forceps. Samples were immediately placed in formalin.
Reproductive history was recorded. Blood samples were collected and when possible were evaluated for plasma testosterone levels, resting thyroxine levels, and serologic assays. Testes were described by color, shape, vasculature, and general appearance. Size was estimated in comparison to the size of the adrenal gland. Body weight was recorded. All biopsy samples were collected in the fall during the non-breeding season, a time that could influence the results, especially in species with a relatively distinct breeding season such as Amazons. Histopathology, as well as Chlamydophila, Aspergillus, Mycobacterium, and Candida, Brucella serology, and plasma Aspergillus antigen analysis were performed by the University of Miami, Division of Comparative Pathology, Miami, FL, USA.
Attempts were made to obtain normal control biopsies; however, biopsy of large active testes is strongly discouraged. In cases where biopsy of large active testes were attempted, spermatozoa and possibly testicular tissue could be observed leaking through the capsule from the biopsy site. In one such case concerning an eclectus parrot that was examined by endoscopy 6 weeks after biopsy, adhesions had formed between the testicle and surrounding viscera.
Study Results
Biopsy samples from a total of 56 birds were evaluated. Five were females, which were mates of 5 males included in this study. These females were biopsied because of suspicious endoscopic findings in the ovaries (ovarian tissue). All ovarian tissue evaluated was considered histologically normal. Eight samples did not contain testicular tissue, and 3 contained histologically normal testicular tissue.
The samples overall were of good quality, although some samples contained no testicular tissue. In these birds, testicular tissue was difficult to obtain due to severe fibrosis of testicular tissue. In some birds, the testes were so hard and the surrounding membranes so thickened that it was difficult to bite into the testicular tissue with the biopsy forceps. In these cases, extensive fibrosis or testicular atrophy was suspected. Because of the thick membranes in these birds, the biopsy often yielded only the surrounding tissue. The remaining 45 birds showed evidence of testicular pathology.
Table 1. Summary of histopathologic findings in birds with testicular pathology.
39 of 45 birds (87%) orchitis (inflammation);
40 of 45 birds (89%) some degree of fibrosis;
1 of 45 birds (2.2%) active spermatogenesis*
4 of 45 (9%) foci of hemorrhage within the testicle biopsy;
4 of 45 (9%) foci of necrosis within the testicle biopsy;
2 of 45 (4.5%) tubular dilation;
2 of 45 (4.5%) tubular mineralization;
1 of 45 (2.2%) changes consistent with tubular degeneration;
* Large numbers of mature sperm were observed histopathologically for this individual. A few mature sperm were observed histopathologically in several other birds, but not in large numbers.
The orchitis was best characterized as mild (55% of cases) to moderate (45% of cases) multifocal, chronic inflammation with fibrosis consisting of lymphocytes primarily (rarely plasma cells) and an average of 43% + 29 (range = 0–95%) fibrosis. The 3 normal testes contained approximately 5% fibrosis. Occasional tubules were characterized by foci of coagulative necrosis, degenerative changes, nuclear pyknosis, or hemorrhage.
A large percentage of testicular samples contained evidence of chronic orchitis. Fibrosis and aspermatogenesis were marked and consistent findings in these punch biopsy samples. Infectious agents were not observed by routine staining. Special stains (Gram stain, acid fast, Wright Geimsa, and silver stain for fungus) were performed on a subset of abnormal and 1 normal testicular sample. No agents were identified by these stains.
A serological survey of 38 of the male birds included in the study revealed 21.1% positive titers to Chlamydophila species, 10.5% positive for Aspergillus antibody, 5.3% Aspergillus antigen, 31.6% positive for mycobacterium antibody, and 28% positive for Candida antibody. These samples were also tested for Brucella canis antibody by the traditional agglutination test used for screening dogs and all samples were negative for antibody.
As an additional method of evaluation of fertility, 37 birds were evaluated for plasma testosterone levels by fluorescent immunoassay. This assay is based on an enzyme-linked immunosorbent assay (ELISA), using illumination rather than radioactivity as an indicator (Access Immunoassay System, Beckman Coulter Inc.). Of these 37 birds, 18 were identified by endoscopy as being abnormal (visible inflammation of testes, reduced size of testes in comparison to adrenal gland). Of these, the average plasma testosterone level was 0.05 ng/ml (0–0.26). In 19 birds identified as normal or active by endoscopy, the average plasma testosterone level was 0.60 ng/ml (0.02–1.89). Reference ranges for domestic mammals at this laboratory are <0.20 µg/dl for non-functional or castrated males and >0.20 µg/dl for intact males.
Discussion
Functional disorders of the testes include atrophy, hypertrophy, hypoplasia, and orchitis/epididymitis.1 Atrophy of the testes can result from a variety of adverse systemic or environmental influences. In mammals, some reported causes include deprivation of pituitary gonadotropins, administration of exogenous androgens or estrogens, ionizing radiation, generalized malnutrition, specific vitamin deficiencies (vitamins A & E), prolonged fever or hyperthermia, certain infectious diseases, denervation, occlusion of spermatic vessels, certain genetic disorders, and certain neoplasms. Hypertrophy and hypoplasia occur rarely and are usually a consequence of endocrine imbalance.1
Orchitis and epididymitis occur frequently in most animal species and may be caused by specific bacterial infections that have a predilection for the testes and epididymus. Brucella abortus in cattle, B ovis in sheep, B suis in swine, B canis in dogs, and rarely Mycobacterium tuberculosis var bovis in cattle. Orchitis may also be caused by a variety of ubiquitous bacteria capable of causing tissue damage in any organ to which they are introduced. These include E coli, Proteus vulgaris, Corynebacterium ovis, Streptococci species, and Staphylococci species. Canine distemper virus may cause orchitis and epididymitis in mature dogs.1
In infectious forms of orchitis, the causative agent enters the testes by several routes including penetrating wounds, hematogenously or from other foci of infection, or reflux of exudates through the vas deferens from infections elsewhere in the genitourinary tract.1 While penetrating scrotal wounds in mammals are a significant cause of orchitis, penetrating wounds in birds would be unlikely, although spread of disease from the respiratory system could occur.
Early lesions of orchitis include hemorrhage, edema, and paremchymal necrosis. This is followed by an influx of inflammatory cells, the intensity and distribution of these cells depending on the virulence of the causative agent. Neutrophils usually appear first and often accumulate in seminiferous tubules. Cellular debris from necrotic spermatogenic and Sertoli cells also desquamate into the tubular lumina. This type of lesion is often seen in brucellosis where caseous necrosis and granuloma formation are characteristic. If spermatozoa were released into adjacent stroma, an intense granulomatous inflammatory reaction could ensue with formation of a spermatic granuloma. This lesion is characterized by an accumulation of large numbers of epitheliod cells around the extravasated sperm. Foreign body giant cells are often present in such granulomas. In mammals, occasionally the infection penetrates the tunica albuginea of the testes and extends into the scrotal sac. The healing phase of inflammatory processes in the testes and epididymus is invariably accompanied by varying degrees of fibrosis and mononuclear inflammatory cell infiltration. Tubules become obliterated or occluded by fibrous connective tissue. Varying degrees of testicular and or epididymal atrophy and occlusion result. Fertility may be normal or severely impaired. Leydig cells proliferate to replace those lost: hence androgen production is not permanently diminished.1
References to orchitis in companion avian patients are sparse and vague.2 Likewise, references to orchitis in poultry are sparse while other causes of male infertility have been studied. McDaniel showed that high environmental temperatures (32.2oC) can contribute to infertility in male chickens when compared to controls. He concluded that fertility in the male bird is influenced more by heat stress than in the female.3,4
Muncher et al studied early age infertility in domestic roosters and associated it with intratesticular retention of sperm. Roosters that are maintained under controlled conditions of high production have peak fertility at 32 weeks of age (96%), but it subsequently declines rapidly to only 5% at 110 weeks despite the fact that roosters can live for about 10 years. Roosters exhibiting this low-fertility syndrome have reduced levels of spermatozoa in the ejaculate. Concomitantly, however, superabundant but apparently normal spermatozoa are found attached to Sertoli cells and, in addition, the seminiferous epithelium fails to show evidence of the regression or atrophy that characterizes both aging non-seasonal breeders and true seasonal breeders during non-reproductive periods. This syndrome of premature low fertility appears to stem from impaired spermiation with resultant retention of spermatozoa by Sertoli cells.5
Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently recognized virus of swine had also been associated with birds. Zimmerman et al exposed several species of fowl to PRRSV (ATCC VR-2402) in drinking water and virus isolation was attempted from feces collected from cages. Based on the duration of fecal shedding of the virus, this experiment showed that Mallard ducks were particularly susceptible to PRRSV. Collectively, these studies show that avian species may be involved in the epidemiology of PRRSV. This was the first report of PRRSV infection in a species other than swine.6
The findings of this study indicate that orchitis in parrots can be associated with infertility and that a possible infectious etiology exists. While the parrots included in this study were primarily in Florida, one author (Clubb) has observed this phenomenon in Central America, South America, the Caribbean, and Europe as well, so it appears to be a ubiquitous problem that is not limited geographically.
While no specific etiological agent was identified, serological analysis suggests a causal relationship. The Chlamydophila seropositive rate in this study of 21.1% is much higher than the 9–10% positive rate found in average random submissions to the University of Miami, Division of Comparative Pathology laboratory. Likewise the Mycobacterium antibody positive rate of 31.6% is much higher than the average random submission positive rate of 5–6%. Aspergillus and Candida serology positive rates are consistent with average submissions. The predominance of lymphocytes rather than plasma cells could suggest a lack of ongoing antigenic stimulation or immune-mediated damage to the testes (B. Ritchie, oral communication, December 2001)
References
1. Jones TC, RD Hunt, NW King. Genital system. In: Veterinary Pathology. Baltimore, MD: Williams & Wilkins; 1997:1149–1222.
2. Joyner K. Theriogenology. In: Ritchie BW, Harrison GJ, Harrison LR, eds. Avian Medicine: Principles and Application. Lake Worth, FL: Wingers; 1994:749–804.
3. McDaniel CD, Bramwell RK, Wilson JL, Howarth B Jr. Fertility of male and female broiler breeders following exposure to elevated ambient temperatures. Poult Sci. 1995;74:1029–1038.
4. McDaniel CD, Bramwell RK, Howarth B Jr. The male contribution to broiler breeder heat-induced infertility as determined by sperm-egg penetration and sperm storage within the hen’s oviduct. Poult Sci. 1996;75:1546–1554.
5. Muncher Y, Sod-Moriah UA,Weil S, et al. Intratesticular retention of sperm and premature decline in fertility in the domestic rooster, Gallus domesticus. J Exp Zool. 1995;273:76–81.
6. Zimmerman JJ, Yoon KJ, Pirtle EC, et al. Studies of porcine reproductive and respiratory syndrome (PRRS) virus infection in avian species. Vet Microbiol. 1997;55:329–336.