Fish Physiology and Biochemistry vol. 8 no. 6 pp 497-499 (1990) Kugler Publications, Amsterdam/Berkeley
Short communication: Induced ovulation of sablefish (Anoplopoma fimbria) following oral administration of des Gly'O-(D-Ala 6 )LH-RH ethylamide I.I. Solar, E. McLean, I.J. Baker, N.M. Sherwood' and E.M. Donaldson Department of Fisheriesand Oceans, Biological Sciences Branch, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, B.C., V7V 1N6; University of Victoria, Department of Biology, Victoria, B.C. V8 W 2 Y2 Keywords: sablefish, Anoplopoma fimbria, gonadotropin-releasing hormone, LHRH analogue, oral intubation, induced maturation, polypeptide absorption, polypeptide protection
Introduction
Materials and methods
The delivery of maturational hormones to fish, via injection, or by implantation of slow-release devices has been successfully employed by the aquaculture industry to induce ovulation in various species of teleost (reviewed by Donaldson and Hunter 1983). However, handling, and a variety of other factors, may inhibit the ovarian response and cause follicular atresia in fish (reviewed by Billard et al. 1981). Thus, an effective method of inducing ovulation without the need of physical manipulation would be extremely valuable to the industry. Various authors (e.g., Amoss et al. 1972) have reported that oral delivery of LH-RH analogues successfully induces ovulation in mammals. Furthermore, Thomas and Boyd (1989), reported that feeding spotted seatrout with LH-RHa injected shrimp provided ovulatory success in this warm water species. In order to further define the feasibility of delivering maturational compounds to fish via the oral route, we delivered a known dose of LH-RHa to sablefish, utilizing an intubation technique. The time-course of absorption of the LHRHa was followed over an eight hour period. Since limited numbers of sablefish were available, it was necessary to devise a means of ensuring maximum possible absorption of the orally delivered LHRHa. Thus, the LH-RHa was co-administered with an antacid and penetration enhancer.
1.4 kg), were Eight captive female sablefish (4.8 maintained under natural photoperiod in a wooden circular aquarium supplied with flowing ambient seawater (range 8.1-8.9°C). Fish were fed herring chunks every second day. All animals were identified by floy tags. Mean oocyte diameter was determined by ocular micrometry. Animals which exhibited a mean egg diameter of > 1.23 mm were selected for LH-RHa intubation. Des Glyl°-(DAla 6 ) LH-RH ethylamide (Syndel Laboratories, Vancouver, B.C.) was dissolved in 1.5 ml 0.65% saline to a final concentration of 1 mg LH-RHa kg- 1 body weight sablefish. To this solution were added 50 mg sodium bicarbonate and 0.5 mg L-lysophosphatidylcholine (Sigma Chemical Co. Ltd., St Louis MO., U.S.A.) which were used to elevate gastric pH and to maximize gastrointestinal absorption, respectively (Tagesson et al. 1980). The LHRHa solution was delivered to lightly anaesthetised (MS222, 95 mg 1- l) fish by oral intubation. Control animals received the identical solution minus the LH-RHa. A second treatment was administered to all fish 11 days later, but the dose of LH-RHa delivered was halved. Blood samples were taken from control and treated individuals immediately prior to intubation and at various intervals thereafter (0.5-8.0 h). Following treatment, fish were weighed at various times to gain an indication of the degree of ovarian hydration. Fish exhibiting a fully distended abdomen and protruding genital papillae
498 Table 1. Response of mature female sablefish Anoplopomafimbria, to oral administration of either LH-RHa in two doses of 1.0 and 0.5 mg/kg or two doses of the saline vehicle. n
Initial wt (kg)
Initial egg diameter (mm)
Dose of LH-RHa (mg/kg)
FInal wt (kg)
Number spawned
Mean latency period (days)
Mean %o fertilization
3
6.1 + 1.6
1.33
0.02
1.5
7.1 + 2.3
3
16.7 + 2.3
la 4
4.8 4.01 + 0.51
1.23 1.17 + 0.02
1.5 -
4.8 4.0 ± 0.53
-
-
22.6 + 10.5 (7.0 + 4.8)b -
a Data for unresponsive animal; b Percent normal development at 4-cell stage.
zT. 200
-
160
120 I
! ! I
80
I
II
%5
!I
I
I I I
40
!
I
II II 0 0
2
4
8 hours
Fig. 1. The time-course of appearance of LH-RHa in mixed arterial-venous plasma samples of sablefish, following a single oral intubation of 1 mg LH-RHa kg- l body weight in buffered saline.
were stripped, their eggs collected and examined for fertilization success. Plasma levels of LH-RHa were determined using the methods of Sherwood et al. (1988).
Results and discussion Mean body mass, initial oocyte diameter, latency period and percent fertilization of control and LHRHa treated sablefish are summarised in Table 1. Between 8-18 d prior to ovulation, treated fish
exhibited only moderate weight gain when compared to control animals. However, 3 days following the second LHRHa delivery, body weight increased in the treated group (p < 0.01). Three of the 4-treated sablefish ovulated and were strip spawned 3-7 days after the second LHRHa treatment (Table 1). Mean percent fertilization success was variable (Table 1), but similar to that obtained when this analogue was delivered by injection (Solar et al. 1987). Test fertilization demonstrated that a high proportion of embryos developed atypically to the 4-cell stage.
499 The data presented in Table I indicate differences in oocyte size (p < 0.05) between control and treated fish. However, since ovulation has been successfully induced following LHRHa injection into smaller individuals, containing oocytes of lower diameter (Solar et al. 1987), it is unlikely that this difference had any effect upon the outcome of the experiment. The individual that failed to ovulate exhibited both the lowest level of circulating LHRHa and its weight remained stable. Thus, it is possible that this animal may have egested a portion of the intubated LHRHa. The time-course of uptake and net plasma presence of LHRHa following oral delivery of 1 mg/kg body wt. is summarized in Fig. 1. LHRHa was detected within 30 min of intubation, peaked at 1 h, and thereafter declined to 33.2% peak presence 7 h later. Control plasma samples were void of detectable LHRHa (ie < 0.001 ng/ml). Thus, the physiological reactions observed in treated animals indicate that the LHRH was present in biologically significant amounts, which permitted the priming of these animals for secondary LHRHa treatment. Although some 10-fold greater quantities of hormone were required to induce ovulation in the sablefish, than required by injection techniques, the results presented demonstrate the feasibility of oral delivery techniques for gastric species of teleost. The results presented also provide the first biochemical evidence for nonapeptide absorption by the teleost gut. Further studies of dose-response
relationships and polypeptide protection methodologies may decrease dose requirement, and the cost of such procedures. Future trials will also test the oral administration of a small dose of analogue as a primer, followed by a larger second dose.
References Amoss, M., Rivier, J. and Guillemin, R. 1972. Release of gonadotropin by oral administration of synthetic LRF or a tripeptide fragment of LRF. J. Clin. Endocrinol. Metab. 35: 175-177. Billard, R., C. Bry and C. Gillet. 1981. Stress, environment and reproduction in teleost fish. In: Stress in Fish. pp. 185-208. Edited by A.D. Pickering. Academic Press, New York. Donaldson, E.M. and G.A. Hunter. 1983. Induced final maturation, ovulation and spermiation in cultured fish. In: Fish Physiology, Vol. IXB, pp. 351-403. Edited by W.S. Hoar, D.J. Randall and E.M. Donaldson. Academic Press, New York. Sherwood, N.M., L.W. Crim, J. Carolsfeld and S.M. Walters. 1988. Sustained hormone release. I. Characteristics of in vitro release of gonadotropin-releasing hormone analogue (GnRHA) from pellets. Aquaculture 74: 75-86. Solar, I.I., I.J. Baker and E.M. Donaldson. 1987. Effect of salmon gonadotropin and a gonadotropin releasing hormone analog on ovarian hydration and ovulation in captive sablefish (Anoplopomafimbria). Aquaculture 62: 319-325. Tagesson, C., L. Franzen, G. Dahl nd B. Westrom. 1985. Lysophosphatidylcholine increases rat ileal permeability to macromolecules. Gut 26: 369-377. Thomas, P. and N. Boyd. 1989. Dietary administration of a LHRH analogue induces spawning in spotted seatrout (Cynoscion nebulosus). Aquaculture 80: 363-370.
Short communication: Induced ovulation of sablefish (Anoplopoma fimbria) following oral administration of des Gly'O-(D-Ala 6 )LH-RH ethylamide I.I. Solar, E. McLean, I.J. Baker, N.M. Sherwood' and E.M. Donaldson Department of Fisheriesand Oceans, Biological Sciences Branch, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, B.C., V7V 1N6; University of Victoria, Department of Biology, Victoria, B.C. V8 W 2 Y2 Keywords: sablefish, Anoplopoma fimbria, gonadotropin-releasing hormone, LHRH analogue, oral intubation, induced maturation, polypeptide absorption, polypeptide protection
Introduction
Materials and methods
The delivery of maturational hormones to fish, via injection, or by implantation of slow-release devices has been successfully employed by the aquaculture industry to induce ovulation in various species of teleost (reviewed by Donaldson and Hunter 1983). However, handling, and a variety of other factors, may inhibit the ovarian response and cause follicular atresia in fish (reviewed by Billard et al. 1981). Thus, an effective method of inducing ovulation without the need of physical manipulation would be extremely valuable to the industry. Various authors (e.g., Amoss et al. 1972) have reported that oral delivery of LH-RH analogues successfully induces ovulation in mammals. Furthermore, Thomas and Boyd (1989), reported that feeding spotted seatrout with LH-RHa injected shrimp provided ovulatory success in this warm water species. In order to further define the feasibility of delivering maturational compounds to fish via the oral route, we delivered a known dose of LH-RHa to sablefish, utilizing an intubation technique. The time-course of absorption of the LHRHa was followed over an eight hour period. Since limited numbers of sablefish were available, it was necessary to devise a means of ensuring maximum possible absorption of the orally delivered LHRHa. Thus, the LH-RHa was co-administered with an antacid and penetration enhancer.
1.4 kg), were Eight captive female sablefish (4.8 maintained under natural photoperiod in a wooden circular aquarium supplied with flowing ambient seawater (range 8.1-8.9°C). Fish were fed herring chunks every second day. All animals were identified by floy tags. Mean oocyte diameter was determined by ocular micrometry. Animals which exhibited a mean egg diameter of > 1.23 mm were selected for LH-RHa intubation. Des Glyl°-(DAla 6 ) LH-RH ethylamide (Syndel Laboratories, Vancouver, B.C.) was dissolved in 1.5 ml 0.65% saline to a final concentration of 1 mg LH-RHa kg- 1 body weight sablefish. To this solution were added 50 mg sodium bicarbonate and 0.5 mg L-lysophosphatidylcholine (Sigma Chemical Co. Ltd., St Louis MO., U.S.A.) which were used to elevate gastric pH and to maximize gastrointestinal absorption, respectively (Tagesson et al. 1980). The LHRHa solution was delivered to lightly anaesthetised (MS222, 95 mg 1- l) fish by oral intubation. Control animals received the identical solution minus the LH-RHa. A second treatment was administered to all fish 11 days later, but the dose of LH-RHa delivered was halved. Blood samples were taken from control and treated individuals immediately prior to intubation and at various intervals thereafter (0.5-8.0 h). Following treatment, fish were weighed at various times to gain an indication of the degree of ovarian hydration. Fish exhibiting a fully distended abdomen and protruding genital papillae
498 Table 1. Response of mature female sablefish Anoplopomafimbria, to oral administration of either LH-RHa in two doses of 1.0 and 0.5 mg/kg or two doses of the saline vehicle. n
Initial wt (kg)
Initial egg diameter (mm)
Dose of LH-RHa (mg/kg)
FInal wt (kg)
Number spawned
Mean latency period (days)
Mean %o fertilization
3
6.1 + 1.6
1.33
0.02
1.5
7.1 + 2.3
3
16.7 + 2.3
la 4
4.8 4.01 + 0.51
1.23 1.17 + 0.02
1.5 -
4.8 4.0 ± 0.53
-
-
22.6 + 10.5 (7.0 + 4.8)b -
a Data for unresponsive animal; b Percent normal development at 4-cell stage.
zT. 200
-
160
120 I
! ! I
80
I
II
%5
!I
I
I I I
40
!
I
II II 0 0
2
4
8 hours
Fig. 1. The time-course of appearance of LH-RHa in mixed arterial-venous plasma samples of sablefish, following a single oral intubation of 1 mg LH-RHa kg- l body weight in buffered saline.
were stripped, their eggs collected and examined for fertilization success. Plasma levels of LH-RHa were determined using the methods of Sherwood et al. (1988).
Results and discussion Mean body mass, initial oocyte diameter, latency period and percent fertilization of control and LHRHa treated sablefish are summarised in Table 1. Between 8-18 d prior to ovulation, treated fish
exhibited only moderate weight gain when compared to control animals. However, 3 days following the second LHRHa delivery, body weight increased in the treated group (p < 0.01). Three of the 4-treated sablefish ovulated and were strip spawned 3-7 days after the second LHRHa treatment (Table 1). Mean percent fertilization success was variable (Table 1), but similar to that obtained when this analogue was delivered by injection (Solar et al. 1987). Test fertilization demonstrated that a high proportion of embryos developed atypically to the 4-cell stage.
499 The data presented in Table I indicate differences in oocyte size (p < 0.05) between control and treated fish. However, since ovulation has been successfully induced following LHRHa injection into smaller individuals, containing oocytes of lower diameter (Solar et al. 1987), it is unlikely that this difference had any effect upon the outcome of the experiment. The individual that failed to ovulate exhibited both the lowest level of circulating LHRHa and its weight remained stable. Thus, it is possible that this animal may have egested a portion of the intubated LHRHa. The time-course of uptake and net plasma presence of LHRHa following oral delivery of 1 mg/kg body wt. is summarized in Fig. 1. LHRHa was detected within 30 min of intubation, peaked at 1 h, and thereafter declined to 33.2% peak presence 7 h later. Control plasma samples were void of detectable LHRHa (ie < 0.001 ng/ml). Thus, the physiological reactions observed in treated animals indicate that the LHRH was present in biologically significant amounts, which permitted the priming of these animals for secondary LHRHa treatment. Although some 10-fold greater quantities of hormone were required to induce ovulation in the sablefish, than required by injection techniques, the results presented demonstrate the feasibility of oral delivery techniques for gastric species of teleost. The results presented also provide the first biochemical evidence for nonapeptide absorption by the teleost gut. Further studies of dose-response
relationships and polypeptide protection methodologies may decrease dose requirement, and the cost of such procedures. Future trials will also test the oral administration of a small dose of analogue as a primer, followed by a larger second dose.
References Amoss, M., Rivier, J. and Guillemin, R. 1972. Release of gonadotropin by oral administration of synthetic LRF or a tripeptide fragment of LRF. J. Clin. Endocrinol. Metab. 35: 175-177. Billard, R., C. Bry and C. Gillet. 1981. Stress, environment and reproduction in teleost fish. In: Stress in Fish. pp. 185-208. Edited by A.D. Pickering. Academic Press, New York. Donaldson, E.M. and G.A. Hunter. 1983. Induced final maturation, ovulation and spermiation in cultured fish. In: Fish Physiology, Vol. IXB, pp. 351-403. Edited by W.S. Hoar, D.J. Randall and E.M. Donaldson. Academic Press, New York. Sherwood, N.M., L.W. Crim, J. Carolsfeld and S.M. Walters. 1988. Sustained hormone release. I. Characteristics of in vitro release of gonadotropin-releasing hormone analogue (GnRHA) from pellets. Aquaculture 74: 75-86. Solar, I.I., I.J. Baker and E.M. Donaldson. 1987. Effect of salmon gonadotropin and a gonadotropin releasing hormone analog on ovarian hydration and ovulation in captive sablefish (Anoplopomafimbria). Aquaculture 62: 319-325. Tagesson, C., L. Franzen, G. Dahl nd B. Westrom. 1985. Lysophosphatidylcholine increases rat ileal permeability to macromolecules. Gut 26: 369-377. Thomas, P. and N. Boyd. 1989. Dietary administration of a LHRH analogue induces spawning in spotted seatrout (Cynoscion nebulosus). Aquaculture 80: 363-370.
