Yutaka Sawamukai

Ovulation Induction with Follicle-Stimulating Hormone Administration in Hawksbill Turtles Eretmochelys imbricata

Abstract: To develop ovulation induction techniques, we investigated the effects of follicle- stimulating hormone (FSH) administration on ovulation in captive hawksbill turtles Eretmochelys imbricata. Porcine FSH preparation was administered by intramuscular injection to four hawksbill turtles with fully-developed follicles (21.6–23.8 mm, measured using ultrasonography) in July or August 2009. Blood samples were obtained and subjected to plasma progesterone measurement, and ovaries and oviducts were observed by ultrasonography just before, and 1–6 days after, FSH administration. One day after the administraion, the plasma progesterone concentration significantly increased from the basal level (<0.01–0.3 ng/mL) to 0.8– 5.1 ng/mL and then dropped to nearly the basal level within 2–4 days. The formation of eggshells was observed two days after FSH administration. These data collectively indicate that in hawksbill turtles FSH administration effectively induces ovulation, progesterone secretion, and egg formation.

Relationships among steroid hormone levels in newborn piglets, birth weight, placental weight, vitality of offspring and litter size

Blood estrone sulfate (E(1) S), estrone (E(1) ), estradiol (E(2) ) and progesterone (P(4) ) in newborn piglets were measured to clarify the relationships among birth and placental weight, vitality of offspring and litter size. First, the association between vital status (normal, weak and stillborn) from 165 newborn piglets of 18 litters and steroid concentrations; second, steroid concentrations from 152 newborn normal piglets and litter size; and third, steroid content in fetal placenta from 50 newborn normal piglets of six litters and litter size, were investigated. In the normal group, the birth and placental weight were significantly higher than those in the other groups. Blood E(1) S levels in the stillborn group were significantly lower, whereas E(1) , E(2) and P(4) were significantly higher compared to the normal group. Blood and placental E(1) S levels in the small litter group were significantly higher than those in the other groups. However, there was no significant difference among the three litter size groups in the levels of steroid hormones in maternal blood. These results indicate that vitality of newborn piglets is related to E(1) S concentration of neonate, to birth weight and placental weight. However, steroid hormone concentrations of newborn piglets were greatly affected by the number of littermates.

Signals of Vitellogenesis and Estrus in Female Hawksbill Turtles

This study reports a viable means of identifying the vitellogenic cycle and limited estrus period in hawksbill turtles for the purposes of developing captive breeding program, based on the combination of blood metabolite parameters (triglyceride, total protein, and calcium levels), feeding status, and ovary condition. Follicle size of two focal captive females showed clear seasonal changes, with major development occurring between March and May (19.0–24.4 mm), and exceeding 25 mm between June and September. Triglyceride, total protein, and calcium levels dropped with follicular development and maintenance (March to October), and then began to rise when follicular retraction occurred from October onwards. The two focal turtles reduced food intake during intensive follicular development (April to May). These findings suggest that blood metabolite parameters and feeding conditions are inferred by the vitellogenic cycle. An additional 10 females exhibiting follicular development were mated with a single male for 7-day period between May and June. Follicle size was measured immediately prior to pairing, and a statistically significant difference in follicle size of 10 females was recorded between the seven failed (20.9 mm) and three successful (23.6 mm) mating events. This indicates follicle development is essential to successful mate and monitoring of vitellogenic cycle may help improve the success rates of captive hawksbill breeding programs.

Induction of Oviposition by the Administration of Oxytocin in Hawksbill Turtles

We set out to develop an oviposition induction technique for captive female hawksbill turtles Eretmochelys imbricata. The infertile eggs of nine females were induced to develop by the administration of follicle-stimulating hormone, after which we investigated the effects of administering oxytocin on oviposition. Seven of the turtles were held in a stationary horizontal position on a retention stand, and then oxytocin was administrated (0.6–0.8 units/kg of body weight; 5 mL). The seven turtles were retained for a mandatory 2 h period after oxytocin administration, and were then returned to the holding tanks. As the control, normal saline (5 mL) was administered to the other two turtles, followed by the administration of oxytocin after 24 h. The eggs in oviducts of all nine turtles were observed by ultrasonography at 24 h after oxytocin administration. The control experiment validated that stationary retention and normal saline administration had no effect on egg oviposition. Eight of the turtles began ovipositing eggs at 17–43 min after oxytocin administration, while one began ovipositing in the holding tank immediately after retention. All turtles finished ovipositing eggs within 24 h of oxytocin administration. This report is the first to demonstrate successful induced oviposition in sea turtles. We suggest that the muscles in the oviducts of hawksbill turtles may respond to relatively lower doses of oxytocin (inducing contractions) compared to land and freshwater turtles (4–40 units/kg) based on existing studies.

Change in Morphology of Spermatozoa from Dismount Semen during the Breeding Season in Thoroughbred Stallions in Japan.

To clarify the physiological changes of sperm morphology in active Thoroughbred stallions during the breeding season, we examined the dismount semen collected from the penile urethra immediately after service. The spermatozoa were analyzed for relationships between the morphology and the stallion’s age or the number of services. Seasonal variation was apparent in the rate of the sperm tail abnormalities, spermatozoa with cytoplasmic droplets, appearance of medusa cells, and sperm head length. Area and width of the sperm head correlated negatively with age (P<0.05). The rate of appearance of medusa cells and the length of the sperm head were positively related to the number of services (P<0.05), and the aspect ratio was negatively related (P<0.01).

Treatment with a High Dose Combination of PMSG/hCG Preparation of Mares Clinically Diagnosed with Ovarian Quiescence during the Breeding Season (Investigation from 1975 to 2000)

A total of 88 thoroughbred mares were diagnosed with clinical ovarian quiescence and subjected to four treatment regimens. Using PMSG, hCG or combinations of both. A high dose combination of 5,000IU PMSG with 5,000IU hCG showed significantly higher rates of marked estrus and ovulation induction (P<0.01) as well as conception rates (P<0.05). In the present study, the administration of a high-dose combination of PMSG with hCG was shown to be an effective treatment of ovarian quiescence in light mares.

Semen Evaluation of Captive Hawksbill Turtles

Abstract This study presented information about the semen evaluation of captive male hawksbill sea turtles, Eretmochelys imbricata, based on an extended 15-mo study using the electro-ejaculation technique. In particular, we demonstrated that hawksbill sperm, which is underactive just after ejaculation, was activated by the presence of urine. The findings are useful for developing optimal semen collection techniques for future artificial insemination programs of hawksbill turtles.

Daily Changes in the Blood Levels of Two Steroids and Other Biochemicals Related to Vitellogenesis and Eggshell Formation during Internesting Intervals in a Captive Female Loggerhead Turtle

Abstract: Blood levels of progesterone (P4), estradiol-17β (E2), calcium (Ca), triglyceride (TG), and total protein (TP) were monitored in a captive female loggerhead turtle throughout a nesting season that included three 13- or 14-d internesting intervals. We observed the turtles ovaries to monitor the appearance of shelled eggs, using ultrasonography, and compared the results of blood analyses and ovary observations. The blood P4 level remained low (<0.2 ng/mL), with the exception of three sharp peaks (1.7, 2.2, and 4.2 ng/ mL) immediately after the initial three nestings. Eggshell formation occurred immediately after the P4 peaks. The blood Ca level gradually dropped to approximately 3.0 µmol/mL during each eggshell formation period but then recovered to approximately 3.7 µmol/mL prior to the next eggshell formation. The blood E2 level fluctuated between 44 and 299 pg/mL, but with two higher peaks (734 and 419 pg/mL) after eggshell formation in the first and second internesting intervals, respectively. During the nesting season, the blood TG level (initially 20 µmol/mL) showed a stepwise drop to 7.6 µmol/mL, while the blood TP level fluctuated between 57 and 69 mg/mL with no distinct peaks. The periodic cycle of P4 and Ca changes aligned with albumen layer secretion around ovulated follicles immediately after nesting, followed by eggshell calcification in the clutch. The relatively small fluctuations in E2, TG, and TP during the nesting season suggested that lipid deposition and preovulatory follicular development were completed prior to the start of nesting.

Intrafollicular Concentrations of Steroid Hormones and PGF2α in Relation to Follicular Development in the Mares during the Breeding Season.

The concentrations of androstenedione, estradiol-17β, progesterone and PGF2α contained in the follicular fluid produced by the follicles in collected ovaries of mares that have had estrous phase during the breeding season were measured and analyzed the relation between the growth stage of follicles and the hormone levels in the follicular fluid. An ultrasonographic diagnostic instrument was used to measure the diameter of the follicles in order to categorize the follicles into three groups the following: 8 small follicles (from 1.0 to less than1.5 cm), 8 medium follicles (from 1.5 to less than 3.0 cm), and 8 large follicles (from 3.0 to 5.0 cm), respectively. The analysis of the follicular fluid in ovaries of estrous mares showed that the concentrations of androstenedione were significantly higher in the medium or large follicles than in the small follicles and the concentrations of estradiol-17β were significantly higher in larger follicles than in the small or medium follicles (P<0.05). The concentrations of progesterone and PGF2α, on the other hand, did not significantly vary regardless of follicluar size. In the follicles within the mare ovaries that have had estrous stage, the concentrations of the hormones related the ovulation, namely androstenedione and estradiol-17β, were higher with larger follicles.