R. E. Larsen; P. T. Cardeilhac
Department of Large Animal Clinical Sciences and Department of Small
Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville,
Leucism is a condition described for white-skinned reptiles that is believed to be caused by a lack of pigment cells (melanocytosis) as opposed to a pigmentosis of the pigment cells caused by an aminoacidopathy. Leucistic alligators do not appear to suffer from the complications that have been reported for albino animals such as low immunocompetence, fibrotic internal organs, poor vision, poor hearing and other problems. Albino animals such as snakes are reported to occasionally have skin problems such as deformed scales that interfere with their locomotion. The objectives of this study were to determine if adult leucistic alligators produce spermatozoa and if sufficient numbers of viable spermatozoa could be collected from the penile groove of the live animal for artificial insemination efforts and for evaluation of semen diluents.
Materials and Methods: Animals
The study subjects were three leucistic adult Alligator mississipiensis collected from the wild as hatchlings (1987) and raised to maturity in indoor pools heated to 31C. Diet was composed of 90% lean nutria supplemented with a protein, fat, vitamin, amino acid, and mineral mix. Each individual received approximately 70 kg of feed per year during the years of the study (1994, 1995). Animals were 244-246 cm in length and 6368 kg in bodyweight. Immobilization was accomplished by intramuscular injection of Valium (0.4 mg/kg) followed by intramuscular injection of succinylcholine (0.3 mg/kg) twenty minutes later (Spiegel, 1984).
The 1994 efforts took place on May 6, the 1995 efforts on May 22. The penis was exteriorized and kept moist with saline. The lumen of the penile groove was catheterized with a 16 ga plastic catheter or with a tomcat catheter pre-filled with semen diluent (Larsen, 1992). Contents of the catheterized duct were aspirated into the catheter. The open distal portion of the penile groove was also scraped with spatulate instruments and plastic tubing to remove mucus and spermatozoa (Cardeilhac, 1982). The products of aspiration and scraping of the penile groove were mixed with 2 ml of semen diluent.
Diluent was composed of 20% v/v egg yolk in 0.75% saline with 1.0% glucose, 500 U/cc potassium penicillin, and titrated to pH 7.0 with TRIS (Larsen, 1984). Osmolality was titrated to 301 mOsM. Diluted semen was maintained at 0°C. No attempt to control the rate of cooling was made. After semen collection, the aspirated duct contents were mixed immediately with diluent in Petri dishes placed on crushed ice. Motility was read on slides warmed to 25C. Semen from each alligator was individually assessed for motility, concentration, and total cell numbers collected, then pooled and diluted to 100 x 106/cc. Concentration was assessed by counts of l:l00 dilutions of extended semen in a hemacytometer. Motility was assessed hourly for 4h and then once daily for 3d. A further dilution of semen from each individual alligator was maintained as a separate sample from the pooled sample. Motility was estimated by observation of multiple cover slipped fields at 400 magnifications.
Results Status of sperm production
Four males were included in the attempt to evaluate reproductive status the first year (1994). One animal failed to respond to the immobilizing drugs sufficiently for safe handling. One animal (#1) had no spermatozoa in the penile groove. Two animals (2S; 4S) produced a small volume of highly concentrated aspirate during the semen collection efforts. This fluid was white, less than 0.1 cc in volume, and was of a density typical of the sediment from centrifugation of body fluids containing cells. The collected material was composed of spermatozoa with so little fluid present that dilution was required for transfer to sample tubes and observation of individual cells under the microscope. The two animals known to produce spermatozoa the first year of the study were evaluated again in 1995. Again, these alligators (2S; 4S) were found to have spermatozoa in the penile groove.
Sperm numbers and motility
In the two animals producing spermatozoa the recovery of cells by the methods utilized resulted in a low of 300 x 106 and a high of 880 x 106. The higher producer of the first year was the lower producer of the second year. Pooled samples from the two alligators allowed a total of greater than a billion cells to be used for artificial insemination of females on the same property. Evaluation of initial motility was sometimes difficult to perform with consistency because of the presence of clusters of immotile spermatozoa that became active during the course of the microscopic examination. Tightly packed, undispersed spermatozoa appeared to be maintained in an inactive state that was changed to motile and active by dilution and separation from clusters. Initial motility was between 80% and 85% in most cases and maintained that level through the first four hours of preservation at 0°C. After three days the motility had noticeably declined, although the results obtained in the first year of the study indicated that three days of transport at 0°C did not reduce viability below what might be required for artificial insemination purposes.
Hypopigmentation of the epidermis is found in fish, amphibians, reptiles, birds and mammals. The extent of pigmentation may vary extensively from no detectable pigment to almost normal coloration. Albinism is a general term used to describe heritable defects of the pigment cells system and is best described for humans in which at least 10 different types have been described.
Two distinct types of hypopigmentation have been described in alligators, true albinism and leucism in which there is reported to be some pigmentation of the skin, which is not translucent as in albino alligators (Atkinson, 1996, pers. com.). Albino alligators have been found to have very short life spans with a number of deformities in the internal organs. The 18 known leucistic alligators appear to have longer life spans and based on necropsy are not affected by the abnormalities found in albino alligators. Leucistic alligators are reported to have the melanocytes essentially absent from the epidermis as is the case with piebaldism in humans which is an autosomal dominant melanocyte condition. Based on results of necropsy and reports of serum globulin levels the leucistic alligators appear to be more similar to normal colored animals than to albino alligators.
Previous studies with Alligator mississippiensis (Larsen, 1984) and Caiman latirostris (Larsen, 1992) have confirmed that saline-based solutions with yolk or milk can maintain crocodilian semen for a period of 3 days or longer at 0°C or refrigerator temperature. Fertile artificial insemination with semen collected from the vas deferens and stored in this way has been achieved in the American Alligator (Cardeilhac, 1988). Freeze-preservation of semen has not resulted in acceptable post-thaw viability. However, transport of semen over long distances could be accomplished with chilled, extended spermatozoa for breeding programs requiring interstate or international cooperation.
The current study establishes that leucistic male alligators raised in captivity can produce spermatozoa at seven years of age and that semen from these animals responds similarly to that from normally-pigmented specimens.
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