Some options to induce oviposition in turtles

A complex interaction of steroid hormones, neurohypophysial peptides, prostaglandins and direct neural modulation is necessary for coordinated oviposition (Guillette et al., 1990b).  Because of this, using exogenous AVT or oxytocin at the incorrect point in the reproductive cycle is relatively ineffective.  We still do not have a complete understanding of how hormones, peptides, prostaglandins and neural modulation interact but some elements have been defined.

After ovulation the corpus luteum secretes progesterone.  The elevated progesterone levels have been shown to inhibit oviductal contractions in vivo in Chelydra serpentina (Mahmoud et al., 1987).  Similarly, exogenous progesterone blocks AVT activity in vitro in Chrysemys picta bellii (Callard, et al., 1976).  Progesterone is also a potent inhibitor of prostaglandin F_2a (PGF) synthesis from the oviduct (Guillette, 1990).  The elevated progesterone levels characteristic of the early post ovulatory phase allows the oviducts to remain quiescent while albumin and shell components are being secreted.

In C. serpentina natural oviposition does not occur until progesterone levels drop after luteolysis (Mahmoud et al., 1984).  A single injection of PGF causes luteolysis and a fall in progesterone levels within 24-30 hours but does not lead to oviposition, so some other factor is probably involved in AVT regulation as well (Mahmoud et al., 1988).

Oviductal tissues of C. picta, Sternotherus oderatus and some other reptiles synthesize PGF.  This synthesis of PGF in the oviducts of reptiles and birds is stimulated by AVT (Guillette, 1990).  AVT, PGF and prostaglandin E₂ levels rise abruptly just before oviposition in two species of sea turtles (Figler et al., 1989, Gross et al., 1992).

In some lizards the administration of a prostaglandin inhibitor can slow or prevent complete oviposition (Guillette et al., 1990a), further supporting the idea that oviposition is the result of a combined direct effect by AVT to increase oviductal tone and a second, indirect, effect produced via PGF that strengthens peristaltic contractions.  A similar pattern occurs in mammals with two distinct oxytocin receptors present in the uterus, one causing increasing tone and the other PGF secretion (Graves, C.R., 1996).  It has also been observed in a variety of species that AVT induced oviductal contractions operate through a different mechanism than PGF induced activity (Cree et al., 1991).

Snapping turtles stressed by capture had elevated levels of estrogen and progesterone for a week after capture (Mahmoud et al., 1989).  Since progesterone blocks the production of PGF and the activity of AVT and oxytocin, the stress of the early stages of captivity can block oviposition via progesterone secretion and/or direct beta-adrenergic stimulation.

It’s been shown in some lizards, birds and mammals that beta-adrenergic stimulation inhibits uterine/oviductal contractions by reducing both PGF production and response.  But, in other lizards, it’s been shown that beta-adrenergic stimulation inhibits the effects of AVT, but not PGF (Cree, et al., 1991).  These effects can be prevented by using a beta-adrenergic blocker (Jones, et al., 1982) and, in some reptiles, a beta-adrenergic blocker alone will induce ovipoisiton (Gross, et al., 1992).  In the lizard Sceloporus virgatus the combination of propranolol and PGF induced normal nesting behaviors (oviposition and nest-guarding) after oviposition (Gross et al., 1992).   A similar process occurs in humans (Quaas, 1985).  One of the treatment options for women with ineffective labor is to use a beta-adrenergic blocker to reduce the peripheral beta-adrenergic response to the stress reaction, thus enhancing the uterine contractions (Sanchez-Ramos et al., 1996).  Therefore, blocking the effects of beta-adrenergic stimulation with propranolol (a commonly used beta-adrenergic blocker) could theoretically enhance the actions of AVT, PGF or oxytocin in turtles.

Another approach to blocking peripheral beta-adrenergic activity is to use an agent that blocks anxiety centrally.  Ketamine is a dissociative anaesthetic very popular in human pediatric medicine because of its safety, anxiolytic action and sedative effects. If the activity of oxytocin or AVT is being blocked by centrally mediated anxiety, ketamine could hypothetically produce anxiolysis and enhance oviposition.  For these reasons propranolol or ketamine were used in conjunction with AVT or oxytocin in parts of this study.

 

Methods and Materials

 

From 1978 to 2006, oviposition was induced a total of 245 times in 13 North American turtle species.  Of the 245 inductions, 195 involved the use of oxytocin alone, 22 used AVT alone, 13 used a combination of oxytocin and ketamine, eight combined propranolol and oxytocin, and seven were given propranolol plus AVT.  Calcium was not administered in any of the cases.

When ketamine or propranolol were used, they were administered intramuscularly (I.M.) at the same time as the oxytocin or AVT was injected intraperitoneally (I.P.).  Since ketamine is cleared by the kidneys (Frye, 1991), I.M. injections were administered into the muscles of the shoulder girdle. This avoids the first pass effect through the kidneys that is characteristic of injections into the pelvic muscles in some reptiles.

Propranolol doses varied between 11-40mcg/kg and were based on the recommended human dose at the time of 14mcg/kg.  There was no cardiac monitoring equipment available to determine the effectiveness of the beta-adrenergic blockade.  Without monitoring equipment it is difficult to know if the beta-adrenergic blockade was adequate to inhibit beta-adrenergic stimulation of the oviducts.

Animals for use in the study came from a variety of sources over the years. Most were obtained from locals living in South Central Pennsylvania that found turtles crossing roads or wandering around their properties or golf courses.  Others came from private collections, known nesting areas, or the edges of waterways in late May to early July.  In the last decade of the study all the subjects came from my own, or other captive colonies, of Trachemys scripta elegans maintained in New Zealand.

Most wild animals were released back to the area of capture.  In cases where that was unknown or the area was in jeopardy (new road development or construction) they were released in the nearest area judged to be relatively safe.  Hatchlings were kept until the yolk sac was completely absorbed and then they were released to the area the female came from or her relocation site.

A 1cc tuberculin syringe combined with a 27 gauge, 1 ¼ inch needle was used for all I.P. injections.  I.M. injections were done with a 26 gauge, 3/8 inch needle.

Before injection the turtle was checked for eggs.  The technique used was to hold the animal carapace up and palpate both inguinal fossae simultaneously by placing the right middle finger (or the middle, fourth and fifth fingers in larger turtles) in the right inguinal fossa while using the index finger and thumb to support the shell.  At the same time, the left hand is positioned in the same fashion in the left inguinal fossa.  By rocking the turtle slowly, and pushing the middle fingers gently in and out, the eggs can be palpated easily.

Before injection the turtle was checked for eggs.  The technique used was to hold the animal carapace up and palpate both inguinal fossae simultaneously by placing the right middle finger (or the middle, fourth and fifth fingers in larger turtles) in the right inguinal fossa while using the index finger and thumb to support the shell.  At the same time, the left hand is positioned in the same fashion in the left inguinal fossa.  By rocking the turtle slowly, and pushing the middle fingers gently in and out, the eggs can be palpated easily.

Four different approaches were used to protect the eggs after induction:  1.  Sling:  Just before the injection a sling was made of two inch wide duct tape and attached to the turtle.  After the injection, the turtle was suspended at a 45 degree angle (head up, tail down) over a moistened towel.  The turtle was kept high enough above the towel to prevent the claws of the fully extended back leg from puncturing the eggs.   Some Trachemys scripta elegans became agitated in the sling and did not oviposit until placed in a tub of water.

2. Tub: In cases where the animal could be continuously observed, the turtle was allowed to expel its eggs in water approximately as deep as three times the carapace height (to prevent damage to the egg).
3. Grid: When there was no concern about damage to the eggs from prolonged immersion, a wide spaced grid was used as suggested by Tucker, et al.  The grid was set 15 cm under the water but allowed the eggs to fall through, beyond the reach of the turtle’s claws.
4. Ramp: Turtles that were injected with ketamine were laid prone on a board slanted at about 20 degrees with their heads up.  From this position the oviposited eggs rolled down the board onto a wet foam pad.

Records were kept of each animal’s species, location and time found, straight line carapace length, drugs used, dosages, time to egg deposition (most cases), number of eggs laid, number of eggs retained, number of injections required and, occasionally, weight.  Straight line carapace length was measured with a dial caliper.  During the first decade, length was used as a basis for determining dosage rather than weight.  This is because weight was influenced by a highly variable egg mass and bladder fluid load, but length was stable.  For this reason weight data is unavailable in some species.

The relative viability of eggs obtained from oxytocin induction versus natural nests was also evaluated.  For this study all 60 nest eggs were obtained at dawn, in the same area, from eight Chrysemys picta picta nests less than 12 hours old (as judged by the residual moisture in the nest plug from the night before).  The superior pole of each egg was marked before removal from the nest to avoid rotation although that was probably unnecessary (Feldman, 1983). In the evening of the same day, in the same location, 14 C. picta were collected on land while in the act of finding a nesting location.  They were injected with I.P. oxytocin (without calcium or other agents) within six hours.  In four cases more than one injection was required 12 hours later.  Sixty two eggs were obtained by injection.  The eggs collected were not defined by clutch but simply divided into two groups by source (either oxytocin induced or natural nest).  All the eggs were incubated in a 50:50 mix by weight of vermiculite and water.  Incubating temperature ranged from 20-38 degrees Celsius as the room temperature varied.

Results

 

Results when comparing the viability of oxytocin induced eggs to natural nest eggs of Chrysemys picta picta

 

Average hatching time was 58 days for both oxytocin induced and natural nest eggs.  Fifty eight (97%) of the 60 nest eggs and fifty seven (92%) of the 62 oxytocin induced eggs hatched.  There was no statistically significant difference (Z = -1.137,   p= 0.256) detected in the hatching rates of the oxytocin induced versus natural nest eggs.

 

Species induced, effective dosages, time to finish laying and success rates using oxytocin alone, in 12 species

 

Terrapene carolina carolina:  Forty two different animals were induced.  Nine were captive specimens.  Length varied from 9.5-15 cm.  Weight varied from 450-700g.   Thirty of the 42 (71%) animals laid all their eggs after the first injection. The most consistently effective first dose was 8-10 units, regardless of size.  Time to finish egg laying after injection varied from ½ to 7 hours.  Six turtles required a second dose of 4-10 units the same day or the next day before they would lay all their eggs.  Three turtles (7%) required a total of 3-5 doses before they laid all their eggs and three (7%) never laid all their eggs despite a total of 2-4 doses over several days.

 

Chrysemys picta picta:  Thirty four different animals were induced.  All were wild.  Length varied from 11.7-15.9 cm.  Twenty two of the 34 (65%) animals laid all their eggs after the first injection.  The most consistently effective dose was 7-8 units regardless of size.  Three turtles required a second dose of 3-10 units the same day or the next day before they would lay all their eggs.  Five turtles (15%) required a total of 3-5 doses before they laid all their eggs and four turtles (12%) never laid all their eggs despite a total of 2-4 doses over several days.

 

Clemmys guttata:  Thirty seven different animals were injected.  All were wild.  Two were injected in successive years for a total of 39 inductions.   Length varied from 9.0-11.6cm.  Weights varied from 150-290g.  During 32 of the 39 inductions (82%) the animals laid all their eggs after the first injection.  The most consistently effective dose was 4-6 units regardless of size.  Time to finish egg laying after injection varied from 2 ½ to 7 hours.  Five turtles required a second injection of 2.5-8 units the same day or the next day before they would lay all their eggs.  Two turtles required a total of three doses each over 2 days before they laid all their eggs.  All turtles laid all their eggs.

 

Trachemys scripta elegans:  Twenty one different captive animals were induced.  There was a total of 44 inductions.  Fourteen turtles had two or more clutches induced with oxytocin;  either successive clutches in one year and/or over several years.  Length varied from 16.2-21.9 cm.  Weight varied from 745-2000g.  During 29 of the 44 inductions (66%) the animals laid all their eggs after the first injection.  The most effective dose for smaller animals was 10-12 units.  For larger animals 14-16 units worked best. Time to finish egg laying after injection varied from 1 ½ to 6 ½ hours.  Five (11%) turtles required a second injection of 10-15 units the same day or the next day before they would lay all their eggs. Seven (16%) required a total of three doses over 2-3 days before they laid all their eggs.  Three turtles (7%) never laid all their eggs despite a total of 3-4 doses.  Two captive specimens that had been kept in aquariums laid double clutches made up of 5-8 hypercalcified eggs and 5-8 normally shelled eggs.

 

Sternotherus odoratus:  Fourteen different animals were induced.  Length varied from 7.0-12.0 cm.  Nine of the 14 (64%) animals laid their eggs after the first injection.  The most consistently effective dose was 5-8 units, regardless of size.  Time to finish laying after injection was 3-4 hours. Two (14%) turtles required a second dose of 3 units (same as initial dose) the same day or the following day before they would lay all their eggs.  Three turtles (21%) never laid all their eggs despite a total of 3-4 doses.

 

Other species:  Smaller numbers (six or less) of Clemmys insculpta, Kinosternon subrubrum subrubrum, Clemmys marmorata, Terrapene carolina major, Terrapene carolina triunguis, Pseudemys rubriventris and Chelydra serpentina were also induced.  Details of the results are in Table 1.  No dosage suggestions were possible for Clemmys insculpta and Chelydra serpentina because of  the poor outcomes.

Table 1.  Outcome of induction using only oxytocin

 

Species	Number of inductions using only oxytocin	Length range in centimeters	Weight range in grams	Suggested oxytocin dosage in units	Suggested oxytocin dosage in units per 100 grams	# injected with the suggested dose	%  success after the first suggested dose	% success with a second dose following the first suggested dose
Terrapene carolina carolina	42	9.5-15.0	450-700	8-10	1.1-2.2	32	81%	88%
Chrysemys picta picta	34	11.7-15.9	250-560	7-8	1.4-2.5	29	74%	83%
Clemmys guttata	39	9.0-11.6	150-290	4-6	2.1-4.0	34	82%	94%
Trachemys scripta elegans	46	16.2-19.4 ————- 19.5-21.9	745-1500 ————– 1200-2000	10-12 ——– 14-16	0.7-1.6 ——— 0.7-1.3	37	76%	84%
Sternotherus odoratus	14	7.0-12.0	200-260	5-8	1.9-4.0	6	100%	n/a
Clemmys insculpta	6	16.0-19.0	Not available	No suggestion	Not available	n/a	n/a	n/a
Kinosternon subrubrum subrubrum	4	8.4-10.0	Not available	3-4	Not available	3	100%	n/a
Clemmys marmorata	4	16-17	Not available	8-10	Not available	2	50%	100%
Terrapene carolina major	2	16.0-16.6	Not available	15	Not available	2	100%	n/a
Terrapene carolina triunguis	2	10.4-11.6	Not available	7-8	Not available	2	50%	100%
Pseudemys rubriventris	1	29	Not available	20	Not available	1	100%	n/a
Chelydra serpentina	1	24	Not available	No suggestion	Not available	n/a	n/a	n/a

 

Effective dosages and success rates using other drugs and drug combinations

The following drugs and drug combinations were also evaluated with smaller numbers of turtles:  oxytocin (suggested dose) + ketamine (<25mg/kg), oxytocin (suggested dose) + ketamine (35mg/kg), oxytocin +propranolol (14-38mcg/kg), AVT (50ng/g) +propranolol (11-14mcg/kg),  AVT (5ng/g),  AVT (25ng/g), and AVT (50ng/g).  Some details and outcomes are shown in Table 2.  The “suggested dose” of oxytocin used in Table 2 was the suggested dose from Table 1 for that species.  Ketamine at a dose of 35mg/kg produced sedation for 3-5 hours so animals must be kept out of the water to avoid drowning until the drug’s effects have completely resolved.

Table 2.  Outcome of induction using AVT or combination therapy

 

Drug or drug combination	# animals induced	# species induced	Percentage success after first dose
Oxytocin (suggested dose) + ketamine (<25mg/kg)	3	3	33%
Oxytocin (suggested dose) + ketamine (35mg/kg)	10	4	90%
Oxytocin + propranolol (14-38mcg/kg)	8	4	50%
AVT (50ng/g) + propranolol (11-14mcg/kg)	7	2	57%
AVT (5ng/g)	4	2	0%
AVT (25ng/g)	4	1	50%
AVT (50ng/g)	14	6	50%

Safety and side-effects

 

Most turtles that were injected were observed and fed for several days or more before release.  There was one death noted among the AVT inductions.  That specimen was a young Clemmys insculpta with an old amputation of a rear leg.  At necropsy an egg was found wedged in a malformed pelvic os.  No other deaths were observed after I.P. injection.  No morbidity was observed among the two Clemmys guttata, nine Terrapene carolina Carolina, and 21 Trachemys scripta elegans (14 injected more than once) that were observed for a year or more after I.P. injection.

Normally, Trachemys scripta elegans in my collection lay eggs every three weeks for a total of 5 clutches per season.  When oxytocin was used for the last clutch of a season the animal induced produced its first clutch of the following year at the appropriate time.   However, when oxytocin induction was used on any given animal earlier in the season, it always delayed the development of the following clutch and usually reduced the total number of clutches that season.  The confounding fact was that the only captive T. scripta that were injected were ones that had retained their eggs at least two weeks after the expected nesting date and/or demonstrated repeated abnormalities in nesting behavior.  However, similar effects have been noted with wild Chrysmys picta in Nebraska (Iverson et al., 1993).

A significant side-effect was observed among a few individuals among the population of captive T. scripta elegans.  Three days to two weeks after laying all their eggs following oxytocin induction the turtles displayed nesting behavior by digging completed nests and then remaining there for 2-3 hours before abandoning the site.  This behavior would only happen once after an induction.  The same animals tended to demonstrate it repeatedly.  Again, the confounding factor was that the only T. scripta that were injected in the collection were specimens that had retained their eggs at least two weeks after the expected nesting date and/or demonstrated repeated abnormalities in nesting behavior.

However, Tucker et al., 1995, observed similar behavior in wild T. scripta, but it only occurred within 12 or 24 hours post induction with oxytocin.  McCosker (2002) observed the same response in two species (three animals) of wild Australian freshwater turtles that were injected with oxytocin.  Despite laying all their eggs after oxytocin induction, the turtles emerged again 15-21 days later to dig nests despite having no eggs to lay.  McCosker (2002) concluded that this behavior “may be attributed to the failure of a normal nesting-related hormonal sequence to occur during artificially induced oviposition.”

This side-effect could pose a potential risk to wild animals treated with oxytocin since they are exposed to multiple dangers while nesting.  Turtles that nest near roadways would be especially threatened.

Discussion

Eggs obtained by oxytocin induction are as viable as eggs obtained from natural nests thus confirming that eggs obtained by induction are useful for a wide range of experimental purposes.  However, this experiment was carried out with oxytocin induced eggs from wild, nesting females.  In the twenty years since that work was done I’ve observed numerous instances where retained eggs obtained by induction from captive animals had hypercalcific shells and never developed.

Dosage recommendations for specific species, using oxytocin alone, are shown in Table 1.  Recommended dosage ranges for oxytocin alone vary from 0.7-4.0 units per 100 grams with considerable variation between species.  With species where 30 or more animals were treated, the success rates with the first injection (using suggested doses) varied between 74% and 82%.  With a second injection that increased to 83-94%.   Tucker et al. had a 91.8% success rate with a first injection of 10 units/kg in wild Trachemys scripta elegans but his definition of success was that the turtle “retained two or fewer eggs” while my definition of success was no retained eggs after the first induction.

It should be noted that five of six Clemmys insculpta responded very poorly to oxytocin.  Other researchers have noted resistance to oxytocin among Chelydra serpentina and Amyda spinifera (J.T. Tucker, personal communication), in certain Asian genera like Cuora, Heosemys, Leucocephalan and Manouria (C. Tabaka, personal communication), and in Chinemys, Heosemys, Melanochelys, Chelonia mydas, and Batagur baska (Ewert, 1985).

The effectiveness of a single dose of oxytocin for many species may not be acceptable, especially for veterinarians with the goal of removing all the eggs with the initial dose.  The fact that some species don’t seem to respond to oxytocin, and the unfortunate side effect of repeated nesting attempts after successful oxytocin induction, highlight the need to find a better method of induction.

Unfortunately, I found AVT alone to be no more effective than oxytocin alone and very difficult to work with.   There is a need to find a combination of easy to use drugs that yields a higher success rate with the initial injection for a wider variety of species.  My results with propranolol combined with AVT or oxytocin were no better than oxytocin alone, but I had no way of confirming the effectiveness of the dosage of propranolol chosen (11-40mcg/kg).  To properly evaluate the potential of beta-adrenergic blockers combined with oxytocin in turtles an experiment would first need to be done to determine the dosage required to reduce the heart rate (It would be reasonable to assume that a dose high enough to reduce the heart rate would also block the adrenergic receptors in the oviducts).  There have been a wide range of beta-adrenergic blockers developed since this work was done so there are now many options other than propranolol, each with varying specificities and durations.

Although only small numbers (13 animals) were involved, there was a suggestion that the combination of ketamine (at a dosage of 35mg/kg) and oxytocin may be more effective than oxytocin alone.  The sedating effects of ketamine also make it physically easy to position the animal for egg laying.  A larger study with this combination could properly evaluate this possibility.

Natural oviposition is complex and, at least, involves the interaction of peripheral beta-adrenergic neurons, AVT and PGF.  Beta-adrenergic activity blocks PGF production and function in some reptiles and AVT activity in others, Some other potential approaches that might take advantage of this relationship would be to use a beta-adrenergic blocker with oxytocin or PGF, or ketamine with oxytocin or PGF.   During natural oviposition AVT increases oviductal tone directly and indirectly strengthens peristaltic contractions via PGF secretion.  This suggests that another treatment option might be to combine oxytocin with PGF.

Any of these five treatment combinations might prove to be more physiologic than oxytocin alone.  A more physiologic induction method has the potential to eliminate the side effects that result from using oxytocin alone and might provide an induction technique that is effective for all turtles.

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Photo captions

 

1-2006:  An illustration of the complexity of the process of oviposition.

 

 

2-2006:  The technique used to palpate for oviductal eggs.

 

3-2006:  The technique of intraperitoneal injection used during this study.

 

4-2006:  Line drawing of an intraperitoneal injection.

 

5-2006:  Chrysemys picta picta laying eggs while in a sling.