Abstract

Crocodilians are the only living members of the ruling reptiles (subclass archosauria) which included dinosaurs, flying reptiles and related primitive vertebrates. The few crocodilian hormones which have been characterized are similar to those found in mammals, birds and other living reptiles. Alligators have been studied by our group as a model for understanding the reproductive physiology and improving the reproductive efficiency of crocodilians. The survival of sperm cells, effect of PMSG on follicle development, effect of GnRH and HCG on ovulation, artificial fertilization of eggs, induction of lay by prostaglandins plus oxytocin and other parameters have been examined. An Al colony of 23 individually-penned females, wild males and zoo animals have been studied over an 8 year period. Hormones derived from mammals (or analogs) have been found to stimulate specific reproductive processes of the alligator. Follicle maturation and ovulation have been induced and the mature follicles fertilized by artificial insemination. Forty four (44) females have laid 1199 eggs (329 fertile) and 222 viable hatchlings have been produced. Nesting rate, clutch size, hatch rate, incidence of deformed eggs, embryonic survival and the percent viable hatchlings indicate that colony management, feeding techniques, clinical procedures, hormone treatments and artifical insemination can be used to improve the reproductive efficiency of alligators without obvious side effects.

Introduction

Modern crocodilia (alligators, crocodiles and gharials) are reptiles that have been grouped into subclass archosauria or ruling reptiles. This subclass includes extinct members such as dinosaurs, flying reptiles and the primitive ancestors of living crocodilia and birds. The American alligator (Alligator mississippiensis) is one of several large modern crocodilia for which a wild harvest has exceeded its reproductive rate. Although no crocodilians are known to have become extinct in the 19th or 20th century, the necessity for protection was clearly evident by the mid 1950's (1). It was apparent that only protection, controlled hunting, farming or ranching would prevent the extinction of many crocodilians. Collection of eggs or hatchlings from the wild and/or controlled hunting would allow the conservation of the species but maintain the industry. Egg collection is the most efficient method of wild harvest since it is estimated that 95% or more of wild eggs never produce an animal that reaches puberty. Many consumers concerned with animal rights and conservation would accept the collection of eggs provided that hatchlings and adult animals are not taken. The cost of providing protected areas, law enforcement, biologists, and administration would probably require funds generated by commercialization of the crocodilian being conserved in most countries.

Any effective plan to meet cost and acceptance would require that a maximum number of commercial animals be produced from the area of conservation. An efficient plan for economic conditions in Florida appears to be an alligator farm that could sustain itself with a breeding colony producing about 1000 hatchlings but receiving 500 hatchlings produced by artificial incubation of eggs collected from the wild (2). Artificial incubation of eggs would increase hatchling yield by as much as twenty fold. Hatchling production by the captive breeding colony would triple product value for the industry. An annual harvest of sufficient eggs from the wild to yield 50,000 hatchlings would support 100 of the economically viable farms described. These farms would generate products with a farm value of $25,000,000 and a retail value of $250,000,000 (10 x farm value). Ten percent (10%) of the farm value ($2,500,000) of the animals produced on these farms could be used to insure survival of the alligator in Florida.

The Aquatic Animal Laboratory and Gatorland Zoo reached an agreement in 1979 to begin the study of reproductive biology and artificial insemination of the alligator. Our objective was to identify the biological parameters which control reproductive efficiency (3). We wished to determine values (factors) for these parameters in order to quantitate their contribution to infertility on the research farm.

Methods

Reproductive Performance Criteria (Female)

Reproductive rate is defined as the mean annual hatchling yield for each member of the population or colony. Reproductive efficiency is the percent of the maximum reproductive rate possible for the population or colony represented by the observed rate. Performance criteria in the present study are used to determine mean values for only the females that have reached puberty in the population or colony.

 Reproductive Performance
The percentage of maximum hatchling yield possible per female (100) represented by the hatchling yield obtained. or Reproductive Performance = (Hatchling Yield/100) x 100 or Reproductive Performance = Hatchling Yield

 Maximum Possible Hatchling Yield

 This value for a single year is considered to be 100 per female. The value is based on the largest clutches of eggs laid, mature follicle counts and anatomy (length) of the oviduct.
Hatchling Yield = Nesting Rate x Clutch Size x Fertility x Embryonic Survival or I x 100 x I x 1 = 100

 Nesting Rate
The percent of females in a colony which lay.

 Puberty The age at which lay begins.

 Clutch Size
The number of eggs laid in a single year.

 Normal Eggs
Eggs in the clutch which are essentially ellipsoid in shape and have a single yolk, albumen, membrane and a hard shell.

 Deformed Eggs
Eggs which have one or more of the normal characteristics missing.

 Damaged Eggs
Normal eggs which are cracked, leaking, or broken.

 Egg Quality
The percentage of viable hatchlings produced from normal eggs.

 Egg Fertility (Fertility)
The percentage of normal and damaged eggs which develop grossly detectable attachments of the chorioallantoic membrane within 10 days of lay when incubated at approximately 31 C.

 Embryonic Survival
The number of fertile eggs which produce hatchlings (survive I day after hatch).

Research Site

Studies were performed on a breeding colony of approximately 250 adult females and a larger number of males in a 5 acre lake (3). A second group of 23 females were housed in individual pens for artificial insemination (AI). The pens were irregular but averaged about 0.1 acre (10 ft x 20 ft) in size (4).

Hormones and Drugs

Hormones used in this study were Pregnant Mare Serum Gonadotropin (PMSG), Human Chorionic Gonadotropin (HCG), oxytocin, estradiol cypionate (ECP), prostin F2 alpha (Upjohn), Gonadotropin Releasing Hormones (GnRH), and the following analogues were used: des-Gly 10, [D-Ala 6]-LH-RH Ethylamide; des-Gly 10, [D-Leu 61 LH-RH Ethylamide; des-Gly 10, [p-phe 61-LH-RH Ethylamide;des-Gly 10 [D-Trp 6]LH-RH Ethylamide; des-Gly 10, [im-Bzi-D-His 61-LH-RH Ethylamide. Chemical restraint of the animals was accomplished with diazepam and succinylcholine chloride as previously described (5). Follicle diameter was determined by ultrasonic imaging of the follicle on restrained females. The methods of semen collection, stimulation of follicle development with PMSG and inducing ovulation with GnRH have been described (4,6). Lay was induced with 30 IU of oxytocin in females primed with 5 mg of prostin F2 alpha. Shelled eggs in females were identified by radiographs of the restrained females. The dates of lay and hatch were determined by daily observation of the nests or incubated eggs.

Reproductive Physiology and Artificial Insemination

Reproductive activity in the alligator is seasonal with one clutch of eggs produced each year. The entire clutch is usually laid at one time and lay follows ovulation by 42 (+/- 4) days. The period from lay to hatch is 65.5 (+/- 4) days. The mean ovulation date for all Al females was day 140 (+/- 9) days or May 20 (May 11 to May 29). Mean ovulation date for 85 wild females that laid on 3 lakes in Florida was calculated to be day 134 or May 13. These results indicate that the reproductive cycle of both wild and captive alligators are similar for animals living in the same geographic region. Alligators and some crocodiles (Crocodylus johnstoni) are called "pulse nesters" because they lay all of their eggs within a few weeks. Other crocodiles (Crocodylus porosus and Crocodylus novaeguineae) nest over a period of a few months (1). The trigger for reproductive activity of alligators (pulse nesters) may be light, temperature or an endogenous synchronization (7). However, the onset of reproductive activity is associated with emergence from torpor. The Florida alligator stops eating in mid November and enters a torpor induced by the cooler temperatures. Emergence from torpor occurs in mid March and accelerated follicle growth begins within 20 days after feeding begins. Keeping the alligator nearer the equator (El Salvidor) changes the breeding season in a manner unrelated to light and temperature but related to rainfall (8). Based on these observations it was concluded that follicle development may be initiated when the major source of metabolic energy is shifted from fat stores to a high volume of nutrients (amino acids) from the gastrointestinal tract. This assumption has promoted the use of high protein diets containing amino acids for breeders emerging from torpor. This technique is currently under investigation but in one trial (23 AI females) the nest rate increased ON), clutch size increased (4%), egg fertility increased (78%) and embryo survival increased (1.5%). Hatchling yield increased 336% over the mean hatchling yield of the AI colony for all years recorded. Other studies in Louisiana have shown that high quality diets increase hatchling yields for alligators (9).

The female alligator reaches puberty at approximately 10 years of age in the wild (9). A 4-year-old female in the AI colony reared on balanced diets in heated houses has been induced to lay fertile eggs. Young females have a membrane that covers the entrance of the oviducts from the cloaca and this membrane has to be broken to allow the introduction of sperm during Al. It is assumed that the membrane is normally broken when the female first lays and that the first clutch will be infertile. The 4-year-old female (99 lbs) laid 17 eggs and produced 8 viable hatchlings. Clutch size increases with age. The mean value for the largest clutch of eggs found on the research farm each year over a 4 year period was 61. Mean clutch size for all nests during this period was 36. Mean clutch size for wild populations in Louisiana have been reported to be 40 (9) and studies on lakes in Florida produce values that range from 31.2 to 46.0 (A. R. Woodward, Florida Game and Freshwater Fish Commission, personal communication). One large female from the AI colony which had been treated with PMSG and estrogen produced a clutch of 85 normal eggs. These results suggest that clutch size can be increased by 25% with hormonal stimulation. Age at puberty can be reduced 60% by management, feeding techniques and hormone treatments. In summary, clutch size was found to be directly related to age and size of the animal but could be increased by diet and hormonal stimulation.

Follicle growth has been followed by ultrasonic imaging (4). Accelerated growth in follicle diameter begins approximately 30 days prior to ovulation with mean follicle volume increasing approximately 4 fold (7cc to 28cc for small females). The doubling time for follicle volume is approximately 15 days during the accelerated growth phase. Hormonal stimulation (ECP and PMSG) during the accelerated growth phase had no significant affect on growth rate. The growth rate of follicles, following lay, slows or stops but begins to increase after emergence from torpor and enters the accelerated phase by approximately 20 days. After 30 days of accelerated growth the rate slows and the follicle is considered to have reached maturity. Emergence from torpor occurs on approximately day 75 or 19 March and the accelerated growth phase has begun by day 100 or 8 April. Some follicles have reached maturation (can be fertilized) by day 130 or May 10 and reach senility (can no longer be fertilized) by day 150 or May 30. Follicles which become senile are reabsorbed or become retained follicles. Females which reabsorb or retain follicles are stressed and subject to bacterial infections.

Although the growth rate of the follicles does not seem to respond to PMSG and/or estrogen stimulation the number of animals which enter a follicular growth phase can be increased. In one trial with the AI colony (isolated females) 75% of the females which had been given PMSG and 16% of the controls entered the accelerated growth phase of follicular growth.

Ovulation did not occur at a high rate (greater than 10%) without hormonal induction in the AI colony. Therefore treatment regimens to induce ovulation were investigated (4). The most effective regimen involved the use of HCG and GnRH. The GnRH analogs appear to be more effective than mammalian GnRH. The des Gly 10, [d-Ala 6]-LH-RH Ethylamide is effective, inexpensive and readily available. Induced ovulation is dependent on mature follicles and results from the best trial (23 animals) was a nesting rate of 44%. Mean nesting rate on the research farm is 22% while mean nesting rate for all years in the AI colony is 25%. Our studies indicate that nesting rate can be increased by at least 22% with diet and hormonal treatment.

Egg fertility has been reported to be 95% for 6000 free-ranging animals in Louisiana (9) and was 83% for 6 lakes in Florida (A. R. Woodward, Florida Game and Freshwater Fish Commission, personal communication). Mean fertility is 69% for the research farm and 27% for the AI colony. The highest fertility reported for a captive colony of more than 30 animals is 80% (9). Diet appears to have a strong effect on fertility. A ratio of 4 females to I male will produce the fertility rates currently reported for captive colonies. Sperm cell survival is a problem in the Al colony because theirs is not continuous breeding as is the case for natural breeding colonies. Fertility records for the Al colony indicate that insemination less than 5 days prior to ovulation produces high egg fertility. A rapid decline in fertility is observed when insemination occurs more than 5 days before ovulation. Low fertility occurs when insemination precedes ovulation by 10 or more days. The longest recorded interval between ovulation and insemination is 18 days. A range of 20 to 500 million sperm cells have been introduced into each oviduct during the AI studies (6). Approximately 125 million sperm cells are considered adequate.

Lay in captivity appears to be stimulated by the presence of nesting material and the possibility for seclusion. Lay is hormonally induced in the AI colony if it has not occurred by 55 days after the last insemination.

The effect of AI and intensive culture on reproductive efficiency is illustrated by a study on a large lake (5000 ha) in central Florida. The lake produces approximately 100 nests per year and has a population of about 2200 alligators. Thirty nine nests were sampled and mean clutch size was determined to be 46 (A. R. Woodward, Florida Game and Freshwater Fish Commission, personal communication). It is assumed that only 5% of all the normal eggs incubated naturally on the lake would produce animals that reach puberty and that 50% are female. The result would be that 115 females reach puberty in 10 years. With a nesting rate of 65% (found in the Louisiana Study) and a clutch size of 30, for the first-time layers, egg production would be 2242 eggs for the 115 females in 10 years.

Artificial incubation of 6 fertile eggs from the lake (0.2% of the 3818 fertile eggs laid) would produce a female that could be induced to lay at 4 years of age. This would give 6 years of lay before her wild-reared siblings began to lay. A mean hatchling yield of 6 per year (6 x 6 = 36 total) would be obtained from this female based on results for the AI colony. Six of the offsprings from the intensively cultured female would lay 2 years and produce 72 hatchlings (6 x 6 x 2 = 72) before the wild females lay. Six offsprings of the intensively cultured female would lay I year and produce 36 hatchlings before the wild females lay. Thus, one intensively cultured female would produce 144 hatchlings in 10 years that would reach puberty and could be released. The 2242 wild eggs would yield 112 hatchlings (5%) that reach puberty. Thus, a single female using technology currently available (intensive culture and an AI program) could approach the estimated natural production of the large lake.

References

1.  Webb G. J. W. and A. M. A. Smith. 1987. Life history parameters., population dynamics and the management of crocodilians. In: Wildlife Management: Crocodiles and Alligators, G. J. W Webb, S. C. Manolis and P. J. Whitehead (eds.). Surrey Beatty & Sons, Chipping Norton, NSW Australia. pp 199-210.

2.  Cardeilhac P. T. 1987. Alligator farming in Florida. Proc. Florida Nutr. Conf., Daytona Beach FL. pp 173-180.

3.  Godwin, F. and P. T. Cardeilhac. 1983 Problems with low reproductive efficiency. Proc. First Alligator Prod. Conf., Gainesville, FL. pp 65-70.

4.  Cardeilhac, P. T. , H. M. Puckett, R. R. DeSena and R. E. Larsen. 1984. Progress in artificial insemination of the alligator. Proc. Second Alligator Prod. Conf., Gainesville, FL. pp 44-46.

5.  Spiegel, R. A., T. J. Lane, R. E. Larsen and P. T. Cardeilhac. 1984. Diazepam and succinyl choline chloride for restraint of the American alligator. J. Am. Vet Med. Assoc. 185: 1335-1336.

6.  Larsen, R. E., P. T. Cardeilhac and T. J. Lane. 1984. Semen extenders for artificial insemination in the American alligator. Aquaculture 42:141149.

7.  Cardeilhac, P. T. and R. E. Larsen. 1983. A summary of the reproductive physiology of the captive Florida alligator. Proc. First Alligator Prod. Conf., Gainesville FL. pp 94-99.

8.  Joanen, T. and A. Ensminger. 1978. The El Salvador alligator project. I.U.C.N. Survival Service Commission Crocodile Specialist Group, Madras, India. pp 1-8.

9.  Joanen, T. and L. McNease. 1987. Alligator farming research in Louisiana, U.S.A. In: Wildlife Management: Crocodiles and Alligators, G. J. W. Webb, S.

10. C. Manolis and P. J. Whitehead (eds.). Surrey Beatty & Sons, Chipping Norton, NSW Australia. pp 329-348.