Histology of the Sea Turtle - Essay Example

Histology of the Sea Turtle BY 397 Areum Son Introduction Different types of reptiles such as sea turtles, crocodilians, and lizards have temperature-dependent sex determination. Temperature-dependent sex determination is a form of environmental sex determination in which the sex of the offspring is determined by the temperatures experienced during the development of the embryo. When the temperatures are higher, a lot or all sea turtles produced are female. However, when temperatures are low, males are produced in larger numbers or all sea turtles produced are male. If the temperatures are at a constant level whereby it can be said they are not high and not so low, sea turtles produce a similar number of females and males. This temperature is known as the pivotal temperature (Janzen and Paukstsis 151). According to Standora and Spotila (711), some molecular mechanisms are responsible for temperature-dependent sex determination. The molecular mechanisms have not been fully established; however, literature suggests some processes that are incorporated into the molecular mechanisms. The first is that the H-Y antigen could be a structuring factor for the heterogenic sex organ. The second is that reversal of sex could take place in males or females that are genotypic. The third indicates that precise sequences of DNA like the satellite DNA part that is located in some snakes could be changed in their phenotypic characteristic like a variable or directing element (Standora and Spotila 711). Sex ratios determined by TSD are important since they are directly related to the reproduction of the population. Sea turtles are also endangered and this is attributed to hunting, pollution, habitat loss and entanglement in nets. Integrating TSD related issues would be beneficial to the recovery of species by conservation programs. Sea turtles are basically targeted for food and shells. Information about TSD is useful in protecting egg hatcheries by choosing an appropriate thermal environment could provide a desired sex ratio (Plotkin, 179). It is important to note that other environmental conditions affect the development of eggs apart from temperature. One of these environmental conditions is moisture. Moisture as an environmental condition affecting the development of sea turtle eggs has been comprehended much better than temperature. If the moisture levels are so low or so high, then, the mortality rate is raised. In the sea, factors resulting to high moisture levels include high tides which force sea turtles to transfer their nests to higher areas. Also, the aeration around the nest is a condition which affects development of eggs. However, it has not been fully determined what levels of aeration are required for optimum egg development (Ripple 24). Sea turtles are heterosexual and adults display sexual dimorphism. These differences are more evident in the final maturation phase as secondary sexual characteristics. Mature males have a large tail which extends beyond the carapace and a strongly curved flipper claw used in holding the females during mating. Mature females have scars and scratches on the anterior edge of the carapace caused by the male during copulation. Generally, the females do not display any secondary sexual characteristics (Poiani 483). The Kemp’s ridley turtle is a sea turtle that is endangered. This could be attributed to the fact that it nests after every two years. Such duration does not give it a cutting edge in terms of reproduction and competition. However, this time difference is catered for by the number of eggs it lays with approximately 270 eggs for each Kemp’s Ridley turtle. The eggs are usually under incubation for one and a half months. Figure 1: Kemp Ridley sea turtle Sex determination occurs at a point during development in temperature-dependent sex determination systems called the temperature or thermo sensitive period. This time is approximately the middle third of incubation in many reptiles with TSD. Most reptiles conform to two patterns, either the Male:Female (MF) in which the males are produced are produced at low temperatures and the females are produced at high temperatures or the Female:Male:Female (FMF) pattern in which the males are produced at intermediate temperatures and females are produced at both low and high temperatures. The male sea turtles are produced at lower temperatures than females. The range of temperatures in which sex ratios change gradually from 100% male to 100% female is referred to as the transitional range of temperatures (TRT) should also be considered. The males will be produced at temperatures below the TRT while the females will be produced at temperatures above that (Ripple 293). There are implications for the reproductive ecology and conservation of a population for sex-ratios from sea turtles (Beckman 68). TSD produces a wide range of sex ratios since the sex-ratios are highly biased therefore can influence the reproductive output in a population. Biased sex ratios could also be attributed to natural selection. Hypotheses have been proposed that males and females have different fitness in a sea turtle population therefore could lead to sex ratios that are biased. Specific sex ratios can be derived in nature through physiological or physical means. For instance, if a beach has temperatures that fall within those needed to produce a given range of sex ratios; it may be suitable for a particular sea turtle population (Verma 122). Materials and Methods The most accurate method for determining the sex of different hatchlings is use of gonadal histology. The following histological techniques were used 1) dissecting and fixing 2) dehydration and cleaning 3) embedding 4) sectioning and 5) staining. 1) Dissecting and fixing This technique involves removal of gonads and subsequent microscopic examination. The whole turtle was dissected out to find gonads and for the quarter shell, the turtle was placed under the microscope to visualize the gonads. After dissection, one of the pair of gonads in each turtle was not tampered with and left in the original jar to prevent it from drying. Information regarding the location, year, number of jars, total number of turtles and turtles per jar was recorded accurately in a ‘Histology Turtle Log’ without any abbreviation. It was recommended that the jars were to be labeled correctly. The jar labels were made from filter paper for each cassette containing a maximum 3 gonads. The gonads was then dissected and labeled. After the making the cassette, it was closed and placed in a beaker with 70% ethanol and this kept the gonads from drying out. 2) Dehydration and clearing Each beaker contained 675ml of clean solution and the hot paraffin storage container filled up to about 2 inches from the rim. The cup containing the dissected cassettes was placed into 9 solutions for 25 minutes of 70% EtOH, 80% EtOH, 95% EtOH, 100% EtOH, Toluene, Toluene, hot paraffin. Finally the cassettes were placed in hot paraffin storage tank inside the paraffin dispenser. The cassette holder was placed in an empty plastic slide box to prevent paraffin from dripping on the floor when transferring the cassette holder from the final hot paraffin container to the paraffin dispenser and the cassettes submerged inside an embedding machine. Using a Bunsen burner, the thin layer of wax was melted off the metal cassette holder. 3) Embedding A paper towel was used to wipe the melted paraffin from the tissue. One cassette was obtained from the hot paraffin storage tank and placed on a paper towel. The cassette was opened rapidly to prevent the gonads form drying up. The small metal well and the plastic ring were held tightly and filled with wax absolutely. After the paraffin solidified, it lost volume and became concave. It is important to have the metal well and the plastic ring complex by preventing the centre of the block from sinking enough to expose the gonads. The gonads were placed at the bottom of the small metal well and oriented so as to be perpendicular to the bottom of the metal well and in the centre of the metal well. A label was placed at the top of the wax and the block set aside to be rigid for a while and left overnight. 4) Sectioning A microtome was used to cut the block into a pyramidal shape. Care was to be ensured so as not to cut the gonads. A ribbon containing gonads with a length of 3 to 6 file length was cranked. Next, slides should be made depending on the sequence of the ribbon and numbered. The slides should be labeled according to what was on the block and the date. The slide was then covered with regular tap water and the ribbons fit onto each slide as much as possible to save the slides. The slides were then placed on a hot plate in a numerical order to keep them dry overnight. These slides were not to be tampered with. In case a ribbon with no gonads visible was obtained, the microtome blade should be changed. Finally, in order to make the block rigid, it was placed in ice water. 5) Staining This procedure has eighteen steps. First, before staining, it is important to ensure that every well contains 250ml of liquid in it and the dates were checked on the slides before staining. The slides were placed in Citrisolv for 5 minutes, then into Citrisolv for 5 minutes, and placed into Citrisolv for 5 minutes, then placed in 100% EtOH for two minutes , 95% EtOH for two minutes, 70% EtOH for 2 minutes, placed in slow running water for three minutes, hematoxlin for 2 minutes, slow running water for 3 minutes, Eosin for 3 minutes, 70% EtOH for 30 seconds, 95% EtOH for 30 seconds, 100% EtOH for 3 minutes, Citrisolv for 5 minutes, and in Citrasolv for 5 minutes. In the final step, pull one slide out of the final Citrisolv and leave the rest of the slides in the Citrisolv. Lay the slide on a paper towel face up and Run a thin line of Permount along the longest length of the slide. Along the long edge of the slide, lay a cover slip down on the slide at angle. If needed, press lightly on the coverslip with the tweezers to remove any air bubbles and ensure the entire slide is covered with Permount. Place the slide onto the tray and double check to see that the cover slip is covering the tissues and has not fallen off of the slide. Citrisolv is useful since it causes minimal shrinkage of tissue and enhances some stains. Works Cited Beckman, Daniel W. Marine Environmental Biology and Conservation. Burlington: Jones and Bartlett Publishers, 2013. Print. Janzen, J F. and Paukstis G.L. "Environmental sex determination in reptiles: ecology, evolution, and experimental designs." Quarterly Review of Biology 66 (1991): 149-179. Web. Plotkin, Pamela T. Biology and Conservation of Ridley Sea Turtles. Baltimore: Johns Hopkins University Press, 2007. Print. Poiani, Aldo. Animal Homosexuality: A Biosocial Perspective. Cambridge: Cambridge University Press, 2010. Web. Ripple, Jeff. Sea Turtles. Stillwater, MN: Voyageur Press, 1996. Print. Standora, Edward A and James R Spotila. "Temperature Dependent Sex Determination in Sea Turtles." Copeia (1985): 711-722. Web. Verma, Ram S. Genetics of Sex Determination. Greenwich, Conn: Jai Press, 1996. Print. Read More