Introduction

In the United States, farming of the American alligator (Alligator Mississippiensis) has become a recognized aquacultural endeavor in Florida, Louisiana and other Southern States. Due to the large numbers of this species in the wild, in zoos, and on farms, the Alligator provides a rich source of materials for investigations in the biology of crocodilians. Both farmed and wild specimens exist near research institutions and provide ready access to live and dead specimens from the wild from captive housing facilities.

Studies In artificial insemination of captive alligators in Florida have been ongoing since 1980. These studies were oriented to identifying causes of infertility in farm and zoo colonies identifying controlling aspects of reproduction in captivity, broadening knowledge of the reproductive physiology of the species, and providing a model for developing procedures to be used in artificial insemination of crocodilians.

Materials and Methods

Alligators utilized over an eight year period for semen studies and artificial insemination were evaluated for the relationship between length of the animal (LN), total testicular weight (TWT), total sperm cells collected from the penile groove and vasa deferential (TC) maximum mobility of sperm cells after two days in semen extender (MM2). Results from 60 animals were available for at least two of these parameters and 50 animals had recorded measurements for all four parameters. Simple correlations between variables were performed using the Statistical Analysis System. The general linear models procedure for analysis of variance was performed for influence of CN and TWT on TC, also using the Statistical Analysis System.

Collection of semen for preservation and artificial insemination studies is by dissection of the genitalia with extraction of the contents of the vas deferens. Males for dissection are available from a number of sources (trapped nuisance alligators, farmed animals harvested for hides and meat) and provide spermatozoa in the numbers necessary for investigations in semen preservation and artificial insemination. The spermatozoa available from each animal was dependent on size with 7-8 foot males providing over one billion cells and males over 11 feet often providing in excess of 10 x 101 cells. Occasional animals with unilateral testicular hypoplasia or failure of normal spermatogenesis were observed.

Sperm cell recovery after dissection of the complete tract (penis, vas deferens and testes) was accomplished by accomplished by placing the vas deferens in a petri dish and scraping 4-5mm lengths of the duct with a scalpel blade and forcing the contents into a drop of semen extender. The stripped portion of the duct was then removed and each subsequent length of duct was treated in similar fashion until the contents of the entire duct was suspended in media. The penile groove was aspirated and scraped, and its contents also placed into medium for measurement of concentration and total number of cells. Recovery of at least one billion cells from the total genital tract was considered to be the minimum for semen from that Minimal] to he used for investigations.

Due to logistical considerations, semen collection was performed the day before artificial insemination was to be done. This presented the possibility of detrimental handling, dilution. and storage effects for 24 hours prior to use. To test the effect of transporting and storing semen in the dissected, cooled, vas deferens, one vas deferens from each of five large males was stripped of its contents in the field 24 hours prior to use, and the other vas deferens maintained on ice for 24 hours and stripped of its contents just before use. Observed semen quality, based on maintenance of motility over time in various extenders, was recorded daily until all sperm motility was lost. Semen collected and extended on the day of dissection (Day 1) was diluted in 6 different extenders with variation in chemical composition and the addition of various levels of cryoprotectants. Semen extended following 24 hour maintenance of the isolated vas deferens on ice was diluted in the same 6 extenders. Motility data for day one was therefore available only for the semen extended on day one. Motility data for day two was available for semen extended on both day one and day two but was not utilized for analysis as one characteristic of alligator semen in extenders has been a tendency to improve in motility after a day or two in storage. Semen from the tract of the animal is so concentrated as to be a semisolid. Dissociation of sperm cells from their clumps initiates motility but there is also an extender-mediated effect on improvement of motility with time. Data from motility estimates of semen in Extenders 1-6 were treated as dependent variables to evaluate the effect of maintaining the isolated vas deferens on ice for 24 hours versus extension of semen immediately following dissection of the reproductive tract.

Results

Correlations between LN, TWT, TC, and MM2 were all significant (p<.Ol) with the exception of TWT to MM2 (Table 1). Analysis of variance for the positive effect of LN on TC was significant (p<.001) regardless of whether the influence of TWT was removed or not. TWT did not have a significant effect on TC following removal of the influence of LN but had a significant (p<.OO1) positive effect on TC when LN was not considered.

Analysis of variance for the influence of day of semen extension (day 1 or day 2) on sperm cell motility on days 3, 6, and 9 was performed with animal (GATOR), day of extension (DE) and diluent (EXT) as class variables and percent motility (MOT) as a continuous variable. For all three observation days (Day 3, Day 6, Day 9) a significant (p<.Ol) effect of day of extension was seen, with storage of the isolated vas deferens at OC for 24 hours producing higher motility than immediate dilution of semen (Table 2), GATOR was significant on day 3 and day 9 (p<.05) but not on day 6. Diluent composition (EXT) was not significant on any day nor was the EXT x DE interaction.

Table 1. Simple correlations between length (LN), total testicular weight (TWT), total sperm cells (TC) and maximum sperm motility on DAY 2 (MM2)

Table 2 Means of percent sperm motility for semen extended on DAY 1 or after 24 hours at OC and assessed for motility on DAYS 3, 6 and 9 (n=30; five alligators, six extenders)

Discussion

These results establish the fairly obvious conclusion that large alligators have larger testes and produce more sperm cells that are available for collection from the penile groove and vas deferens than smaller alligators. They also suggest that larger animals which have more cells available for collection produce semen which maintains higher motility when diluted in semen extenders. This may be a result of handling effects which tend to favor manipulation of larger volumes and larger numbers of collected spermatozoa.

Results are given from experiments to compare motility maintenance of spermatozoa in diluents when semen was extended immediately after dissection of the reproductive tract or when semen extension took place after keeping the dissected tract at OC for 24 hours. Extending semen immediately after dissection does not appear to have advantage over maintaining the dissected tract at OC for 24 hours. During the year of this study, the conditions under which the experiment was performed gave an advantage to dilution of semen after 24 hours.

The reasons for this are not clear. The immediate collection of semen was done in petri dishess placed on ice, so temperature should not have been a factor. Sperm cells are stored in the vas deferens as tightly packed immotile cells. Dilution and acquisition of motility are likely to be detrimental to the long term storage of semen. The extra 24 hours of dilution and metabolism suffered by the DE=l semen may have given the DE=2 semen a 24 advantage before initiation of degenerative changes. Although this was not analyzed statistically, the decline of motility of DE=l semen appeared to be greater than the 24 hours difference between the two groups could explain.

Storage of semen for 24 hours in the dissected and isolated was deferens offers investigators a number of benefits. Often, trapped and hunted animals must be dissected ill the field for tissue to be available for scientific studies. The ability to transport or mail tissue on ice to a laboratory for more carefully controlled manipulations depends on evidence that detrimental autoanalysis does not take place during the period of transport. This study suggests that in the case of spermatozoa from the vas deferens 24 hours of storage at OC within the vas deferens is not detrimental to motility then immediate collection and dilution. A direct comparison of fertility of semen under DE= 1 versus DE=2 conditions has not been possible. Motility is not synonymous with fertility but is assumed to be related.

References

1.  Cardeilhac, PT, HM. Puckett, RR Desena and RE Larsen, 1982. Progress in artificial insemination of the alligator. Proc. Second Alligator Prod. Conf., Gainesville, FL. pp. 44-46.

2.  Cardeilhac, PT, Larsen, RE, F. Godwin, M, Godwin and DK Peters, 1988. Reproductive Biology and artificial insemination of the American alligator. Proceedings, Ann Mtng Int. Assoc. Aquatic Anim. Med. Vol 19: 179-185.

3.  Larsen, RE, and Cardeilhac, 1983. Collection and maintenance of semen for artificial insemination. In: Proc. Ist Annu. Alligator Production Conference, 12-13 February Gainesville, FL, pp.

4.  Larsen, RE, Cardeilhac, PT and Lane, TJ, 1984. Semen extenders for artificial insemination in the American alligator. Aquaculture 42:141-149.