Abstract

Semen was collected post-mortem from 19 male alligators (Alligator mississippiensis) by dissection of the genitalia. In 17 of 19 alligators, adequate numbers of spermatozoa (> I X 10) were collected for semen preservation and artificial insemination studies. Semen extended in solutions containing a combination of egg yolk and milk, rather than either of these constituents alone, were superior for long-term liquid storage. The addition of either penicillin with dihydrostreptomycin or gentamicin in combination with yolk and milk enhanced the longevity of sperm cells, as compared to maintenance of motility in solutions without antibiotics. Both glycerol and DMSO were detrimental to sperm cell motility at concentrations of 5% and 10%.  DMSO was less detrimental than glycerol.

Introduction

Artificial insemination has been utilized successfully in the American alligator for four years beginning in 1981 (1). However, the problems associated with reproduction in captivity--a low rate of nesting and egg-laying among the females, and poor fertility in the eggs laid--have made assessment of semen handling and insemination techniques difficult, due simply to low numbers of fertile matings. As a result, the evaluation of semen diluents, or extenders, has taken place in vitro with the selection of semen handling practices dictated by the best results from semen motility and longevity testing. Obtaining adequate numbers of sperm cells for study was the first hurdle in the process.

Electrical stimulation of the cloaca appears to be of only minimal value for inducing emission of spermatozoa from the penis of the alligator in numbers adequate for artificial insemination or semen preservation studies. Current semen collection efforts have concentrated on dissection of the male genital tract and recovery of spermatozoa from the isolated vas deferens (1). This method has provided large numbers of motile sperm cells suitable for successful artificial insemination and basic Studies in semen extender composition for semen storage and uryopreservation. In the following study, an assessment is made of sperm cell numbers available by a post-mortem collection technique. Various semen extender compositions are compared for sperm cell maintenance qualities.

Materials and Methods Sperm Cell Recovery

Nineteen alligators sacrificed by zoos, farms, and trappers during April and May of 1981, 1982, and 1983 were killed by gunshot into the cervical spinal cord just caudal to the cranium. The genital tract was immediately dissected intact, including the testes and penis; the vasa deferens and epididymides were isolated from the caudal pole of the testes to the junction of the penile crura. The right and left vasa deferens were isolated and separated into individual petri plates. Sperm cells within the duct were removed by scraping a 4-5 mm length of 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 treated in similar fashion until the contents of the entire duct were suspended in media. The total volume of media was measured and a small aliquot diluted 1/100 or 1/1000 for measurements of sperm cell concentration in a hemocytometer. The penile groove was aspirated and scraped and its contents also placed into medium for measurements of concentration. Recovery of at least one billion sperm cells from the total genital tract was considered to be the minimum useable for investigations involving semen.

Semen Extenders

The genital tract from six alligators provided spermatozoa to test twenty-four different extenders. A 75 mm section of the proximal vas deferens was taken from each alligator and 3 mm sections were stripped directly into two milliliters of the extender to be tested. Each extender was titrated to pH 7.1 and osmolality of 0.310 Osmoles (before addition of glycerol or DMSO; extenders #17-24). Lipoprotein sources used were raw egg yolk and powdered skim milk reconstituted to 0.310 Osmoles. They were utilized individually or in equal parts to compose 10%, 20%, or 30% of the mixture on a vol/vol basis. Reagent grade glycerol (Glycerin, Sigma Chemical Co., St. Louis, MO) or DMSO (Dimethly Sulfaxide, Mallinckrodt, St. Louis, MO) was utilized in extenders #17-24 as potential colligative agents for freeze protection. They were added as 5% or 10% of the solution on a vol/vol basis after addition of yolk or milk. Antibiotics utilized (extenders #10-16) were sodium penicillin (Penicillin G Sodium for injection, E.R. Squib and Sons, Inc., Princeton, NJ) in combination with dihydrostreptomycin sulfate (Streptomycin Sulfate, Pfizer, Inc., New York, NY) or gentamicin sulfate powder (Garamycin Diagnostic Reagent, Schering Corp., Kenilworth, NJ). Extender #16 utilized gentamycin injectable solution (Gentocin Schering Corp., Kenilworth, NJ) titrated to pH 7.1 with NaHCO₃. Table I tabulates the components of each extender. Extenders were mixed at 0.320-0.130 Osmoles and sterile distilled water was added to bring final osmolality to 0.310 Osmoles. A zwitterionic buffering system which had previously demonstrated promise in low-temperature liquid storage of semen was employed (1,3). All solutions, except #16, contained 22.2 g/L BES ((N,N-bis-(2-hydroxymethyl)-2-aminaethane sulfonic acid)), 6.3 g/L tris (Hydroxymethyl aminomethane), and 28.5 g/L glucose before addition of yolk, milk, DMSO, or glycerol. Solution #16 contained 54 g/L glucose before addition of milk, but contained no BES or tris.

After initial motility was assessed, the samples were cooled to 5 C. Motility was assessed daily by phase contrast microscopy at 25 C until all motility was lost. Motility maintenance between extenders was assessed by Duncans new multiple range test for differences between group treatment means. The data tested consisted of pooled means of motility over a 9 day treatment period.

Results Sperm Cell Recovery

Table 2 summarizes the recovery of sperm cells obtained from the vas deferens of all alligators and from the penile groove of nine of the animals. Cell numbers were related to alligator size and to season, but were not consistent between alligators. In general, sexually mature male alligators contained in excess of 1.5 x 10⁹ in calls within the vas deferens when collected between mid-April and the end of May. While sperm cell numbers were lower in smaller animals than for the larger alligators, the morphology and motility were similar to spermatozoa obtained from larger animals. Four alligators were specimens culled from zoos for missing limbs and debilitating injuries not related to the reproductive tract. In spite of this, all were undergoing active spermatogenesis and two had sperm cell numbers in the same range as healthy wild animals.

Five of the nine animals from which penile spermatozoa were collected provided over one billion cells from that source. There was a wide variation in the numbers of penile spermatozoa and unless sperm cells were present in high concentration, semen tended to be difficult to collect and utilize for physiological studies or artificial insemination. In animals with low concentration of penile spermatozoa, the cells were collectable only in clumps of mucus and had lower motility when diluted than deferential spermatozoa from the same animal. Using a target of 1 x 10⁹ cells as a minimum acceptable yield, 17 of 19 alligators provided sufficient numbers of normal appearing, motile spermatozoa for potential use in semen handling and artificial insemination studies.

Semen Extenders

The mean percent motility of incubated spermatozoa in the semen extenders, not containing DMSO or glycerol, is summarized in Table 3. The evaluation of sperm motility in extenders was complicated by a progressive acquisition of motility over 2-3 days with some samples, and a reduction of motility over the same period in others. For extenders # 1-15, in most individual samples and in all of the means of individuals within an extender group (with the exception of extender #1), there was an increase in motility from day one to day two. In an occasional sample, motility did not peak until day four. The increase in motility from day 1 to day 2 was statistically significant (p < 0.05) for the daily pooled means. A small advantage to the use of 20% protein source (yolk/milk), rather than concentrations of 10% and 30%, seemed indicated by motility over the first few days, but did not maintain a statistically significant advantage over time. On day two, all four extenders containing antibiotics with milk or a combination of yolk and milk (#12-15) had mean motilities higher than any other extender. In samples both with antibiotics and without antibiotics, the combination of yolk and milk was superior to yolk or milk alone in maintaining motility for longer than four days. At day 9, only samples #14 and #15 with a combination of yolk, milk, and antibiotics had mean motilities of over 10%.

Bacterial growth in those samples containing no antibiotic was associated with loss of motility. Since collection of semen was done in a clean but not sterile manner, it was assumed that all samples were contaminated. However, many samples remained essentially free of bacteria for 6-7 days, while others were rapidly overgrown.

Initial motility in extenders containing glycerol was zero. In occasional samples with 5% glycerol, a small amount of movement by cells was initiated after 24 hours. No motility was seen in any sample containing 10% glycerol. Initial motility was present at both 5% and 10% DMSO. However, motility increased after 24 hours in 5% DMSO and decreased following incubation in 10% DMSO

Discussion

Rrecent artificial insemination studies suggest that an adequate insemination dose for semen placed directly in the oviducts may require at least 200-300 million cells. With the low percentage of females expected to successfully reproduce in captivity in any one year (4,5), the production of a single fertile clutch requires the insemination of a number of females and the use of many billions of sperm cells. By using spermatozoa collected from the isolated vas deferens, it has been possible to pool sufficient numbers of cells to initiate studies in artificial insemination and semen preservation in the American alligator. Successful artificial insemination with fertilization and live young have followed (6). It has been noted in this study that occasional animals may contain sperm cells numbering in the billions within the groove of the penis. In such cases, it would be possible to collect numbers adequate for these studies from the live animal.

While there are many problems to be solved in the development of effective semen extenders for crocodilians, there are certain conclusions which can be made from the studies to date. First, both yolk and milk are effective in maintaining sperm cell motility for up to a week, and the combination of yolk and milk seems to provide superior protection than either constituent used singly. Second, while antibiotics (penicillin with dihydrostreptomycin and gentamicin) may not enhance survival directly, they apparently do not inhibit survival and appear to be of value in preventing the adverse effects of bacterial contamination. Third, both glycerol and DMSO have severe inhibitory effects on motility maintenance at concentrations of 5 and 10 percent, but with DMSO causing much less inhibition of motility then glycerol. Studies in freeze-preservation for semen of this species may require lower levels of glycerol or DMSO if motility is to be used as a parameter of assessment.

The American alligator provides an ideal model for studies in artificial insemination and semen physiology of crocodilians in general. There are large numbers of alligators in captivity on farms and in zoological exhibitions. There are also large numbers in the wild; a number of which are harvested by controlled, legal hunts. These three sources--farms, zoos, and wild trapped alligators--provide a wealth of biological material for both anti-mortem and post-mortem studies. The information obtained provides a valuable information base for extension to other farmed crocodilian species and to endangered species for which propagation in captivity may provide the sole means of survival.

Acknowledgements

This research was supported by a grant from Sponsored Research, University of Florida. An expanded version of this manuscript will be published in Aquaculture.

References

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