# Stimulation of spermation by LHRH analogue in carp

F. Takashima, Claudine Weil, Roland Billard, C.W. Crim, Alexis Fostier

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F. Takashima, Claudine Weil, Roland Billard, C.W. Crim, Alexis Fostier. Stimulation of spermation by LHRH analogue in carp. Nippon Suisan Gakkaishi, 1984, 50 (8), pp.1323-1329. hal-02728614

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<figure><img src="image_1.png" /></figure>
# Stimulation of Spermiation by LHRH Analogue in Carp

Fumio TAKASHIMA,*¹ Claudine WEIL,*² Roland BILLARD,*³

Laurence W. CRIM,*⁴ and Alexis FOSTIER*²

(Accepted December 15, 1983)

The effects of daily administration of LHRH analogue (D-Ala₆-desGly₁₀) LHRH ethylamide in NaCl or propylene glycol for five days was studied on spermiation, plasma gonadotropin and 11-ketotestosterone levels. Daily injection of 10 µg/kg body weight of LHRH analogue in 0.7% NaCl increased the volume of collectable milt, the number of spermatozoa, and serum gonadotropin and 11-ketotestosterone levels. Daily injection of 1 µg/kg in NaCl or 1 µg/kg and 10 µg/kg in propylene glycol did not stimulate these responses. These results indicate that LHRH analogue can stimulate the secretion of GtH from pituitary gland which in turn stimulates androgen secretion and the spermiation in the testis.

Mammalian LHRH or its synthetic analogues stimulate ovulation in ayu *Plecoglossus altivelis*,¹⁾ Chinese carp,²,³⁾ goldfish,⁴⁾ medaka *Oryzias latipes*,⁵⁾ plaice,⁶⁾ goby⁶⁾ and tench.⁷⁾

In male, although the elevation of plasma gonadotropin hormone (GtH) after LHRH or LHRH analogues injection, are reported in carp,⁸,⁹⁾ brown trout,¹⁰,¹¹⁾ rainbow trout,¹²⁾ goldfish,¹³⁾ only few trials have been conducted to induce spermiation except in chinese carp,³⁾ pike⁴⁾ and landlocked salmon¹⁵,¹⁶⁾ and some preliminary work in carp.¹⁷⁾ Thus in the present experiment, the effect of a LHRH analogue[(D-Ala₆-desGly₁₀)LHRH ethylamide](LHRH-a) on spermiation was examined and plasma GtH and 11-ketotestosterone levels were measured.

It is also pointed out that the effects of both LHRH and its analogues are different with the methods of administration. For instance sesame oil¹⁸⁾ or Freund adjuvant⁶⁾ were recommended as a vehicle because they improve the effectiveness of the peptides by allowing a slow release of the hormone into the blood. Therefore, besides saline solution, propylene glycol was used in this experiment as a vehicle of LHRH-a.

## Materials and Methods

The experiment was carried out at the Lab.

Fish Physiology, INRA from June to July in 1981. Six-year-old males (average body weight 1.6 kg) which gave small amount of milt by hand stripping were selected. They were divided into 6 groups (each consisting of 7 animals) and injected with the following substances.

Group a: 0.7% NaCl (0.1 ml/kg body weight)

Group b: LHRH-a[(D-Ala₆-desGly₁₀)LHRH-ethylamide](Sigma) in 0.7% NaCl at the dose of 1 µg/kg b.w.

Group c: LHRH-a in 0.7% NaCl at the dose of 10 µg/kg b.w.

Group d: propylene glycol (0.1 mg/kg b.w.)

Group e: LHRH-a in propylene glycol at the dose of 1 µg/kg b.w.

Group f: LHRH-a in propylene glycol at the dose of 10 µg/kg b.w.

During the experiment, the fish were kept in 100 l tanks with running, recirculated water. Average water temperature was 15.7-16.0°C.

Injections were administered intraperitoneally and repeated daily for 5 days. Every morning the milt was expelled by hand stripping before injection and its volume was measured. At the same time 1 ml of the blood was taken from the caudal vasculature for GtH and 11-ketotestosterone measurements.

After the final injection (5th), milt and blood samples were collected for another 2 or 3 days.

<footnote>*¹ Laboratory of Fish-Culture, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108, Japan (陸島史夫:東京水産大学).</footnote>

<footnote>*² Institute National de la Recherche Agronomique, Laboratoire de Physiologie des Poissons, Campus de Beaulieu 35042 Rennes, France (フランス国立農学研究所).</footnote>

<footnote>*³ Institute National de la Recherche Agronomique, Department Hydrobiologie, 78350 Jouy en Josas, France (フランス国立農学研究所).</footnote>

<footnote>*⁴ Marine Science Research Laboratory, Memorial University of Newfoundland, St. John's, Newfoundland A1C 5S7, Canada (ニューファンドランドメモリアル大学).</footnote>
<figure><img src="image_2.png" /><figcaption>Fig. 1. Changes of volume of the milt per body weight (mean±SD) expelled by hand massage. Before injection average amount of milt was about 0.01% or less in every groups. After first injection the amount of milt from some individuals receiving 10 µg/kg b.w. of LHRH-a in 0.7% NaCl solution was increased.</figcaption></figure>

The number of spermatozoa was estimated by a densitometric method. Namely, 2 µl of stripped milt was diluted with 5 ml of 0.7% NaCl solution, and its optical density was measured at 410 nm.

The number of spermatozoa (y, ×10⁹) in 1 ml of milt was calculated from the following expression as previously determined; y=66.35x-0.134 (x: optical density at 410 nm).*

Serum GtH was measured by a double antibody radioimmunoassay using a purified preparation of gonadotropin as described by BRETON *et al.*,¹⁹⁾ and 11-ketotestosterone was measured by the method established by FOSTIER *et al*.²⁰⁾ The Cochran-Cox t-test was used to test significance of the results.

## Results

### *Volume of Milt*

The milt expelled before hormone injection was about 0.01% or less as expressed by percentage of the body weight. During the experiment, the amount of milt in the control groups which received

0.7% NaCl (Group a) or propylene glycol (Group d) was usually lower than this value. On the other hand, in the males which received 1 µg/kg of LHRH-a in NaCl solution (Group b) the collectable volume of milt was 0.02% to 0.03% of the body weight the day after each injection (Fig. 1). The quantity of milt gradually increased day by day, and cumulative amount exceeded a volume of 0.03% of the body weight after the 4th injection.

The animals injected with 10 µg/kg of LHRH-a in NaCl solution (Group c) responded well to this hormone and a large quantity of milt was expelled every day. Average amount of the milt per body weight in this group was 0.05-0.07% and 0.14-0.17% after the 1st and 2nd injection, respectively. The response of LHRH-a was different among individuals, and average ratio had increased day by day in the Group c reaching 0.29-0.30% after the 5th injection (Fig. 1). Some individuals responded well and expelled the milt over 0.3% in each day. Cumulative amount of milt in Group c exceeded 0.2% after 2nd injection and reached about 1% after 5th injection. In one individual

<footnote>* unpublished data.</footnote>
<table><caption>Table 1. Changes of concentration (upper, ×10⁹/ml) and total number (below, ×10⁵/g b.w.) of spermatozoa in the milt after injection of LHRH-a (mean±SE). Figures in the parenthesis indicate the number of individuals</caption><thead><tr><th rowspan="2">Group Administration</th><th rowspan="2">Before injection</th><th colspan="5">Times of injection</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td>1 0.7% NaCl</td><td>(4) 43.4±6.1<br>4.3±0.6</td><td>(5) 39.1±4.8<br>5.2±2.4</td><td>(7) 38.6±6.0<br>4.6±2.1</td><td>(2) 30.4±3.2<br>4.4±1.6</td><td>(1) 36.2<br>3.6</td><td>(1) 33.7<br>3.4</td></tr><tr><td>2 1 µg/kg, NaCl</td><td>(6) 36.5±2.1<br>4.8±1.7</td><td>(7) 29.7±3.5<br>5.6±2.8</td><td>(7) 30.8±3.5<br>7.9±3.0</td><td>(7) 27.4±7.3<br>5.3±2.2</td><td>(7) 29.5±5.6<br>9.2±5.8</td><td>(6) 27.9±4.9<br>6.4±4.2</td></tr><tr><td>3 10 µg/kg, NaCl</td><td>(6) 40.3±5.3<br>4.7±1.8</td><td>(7) 25.4±2.1*<br>18.5±15.6</td><td>(7) 28.0±5.5<br>38.2±5.0**</td><td>(7) 24.7±1.8<br>51.6±4.4**</td><td>(7) 26.0±3.8<br>57.7±17.7</td><td>(7) 24.4±1.6<br>61.6±45.4</td></tr><tr><td>4 propylene glycol</td><td>(6) 34.5±8.3<br>4.7±3.2</td><td>(4) 34.5±4.0<br>5.3±2.4</td><td>(5) 37.5±5.8<br>7.5±9.0</td><td>(1) 26.0<br>10.5</td><td>(1) 36.2<br>3.6</td><td>(2) 43.6±0.8<br>4.4±1.3</td></tr><tr><td>5 1 µg/kg, p.g.</td><td>(3) 43.6±0.8<br>3.6±1.4</td><td>(4) 32.8±8.1<br>3.3±0.9</td><td>(7) 35.2±8.2<br>3.5±0.9</td><td>—<br>—</td><td>—<br>—</td><td>—<br>—</td></tr><tr><td>6 10 µg/kg, p.g.</td><td>(5) 42.6±2.9<br>4.3±3.4</td><td>(6) 34.5±1.3<br>6.8±3.2</td><td>(6) 33.2±4.0<br>10.2±7.3</td><td>(6) 26.9±5.4<br>6.7±3.6</td><td>(4) 28.9±4.9<br>6.5±4.8</td><td>(7) 34.2±6.5<br>13.1±2.8</td></tr></tbody></table>

<footnote>* Significantly different from the control by t-test; *P*<0.05.</footnote>

<footnote>** Significantly different from the control by t-test; *P*<0.001.</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 2. Changes of serum GtH (mean±SD) after injection of LHRH-a. By the administration of 10 µg/kg b.w. of LHRH-a in 0.7% NaCl solution serum level of GtH in some individuals was increased. *; Significant at *P*<0.05, **; *P*<0.001.</figcaption></figure>

the cumulative amount of milt expelled was 1.3% after 3rd injection, and 2.8% after 5th injection.

Collectable milt volume in males injected with 1 µg/kg of LHRH-a in propylene glycol (Group e) did not differ with that of the control and even showed some tendency to decrease after 3rd stripping. On the other hand, the amount of milt from the males receiving 10 µg of LHRH-a in propylene glycol (Group f) was not statistically higher than that noticed in the controls. Cumulative amount of milt in this group was relatively less and almost identical to that of the fish injected with 1 µg/kg in 0.7% NaCl.

In this experiment, the spermiation in each group was also checked after the final injection for successive 4 days. The collectable milt volume in the groups which received LHRH-a at the dose of 1 µg/kg or 10 µg/kg in saline solution as well as 10 µg/kg in propylene glycol were all decreased and reached less than 0.01% to the body weight (Fig. 1).

Three days after final (5th) injection, two individuals were selected from Group c and injected with 10 µg/kg of LHRH-a in saline. The amount of collectable milt increased again in this case and was 0.26±0.20% to the body weight. This phenomenon clearly demonstrated that LHRH-a possessed repeated activity to increase collectable milt volume in carp.

*Number of Spermatozoa*

The average number of spermatozoa in collected milt of all individuals before injection was 38.8±7.3×10⁹ per ml. The concentration of the spermatozoa in all groups showed some slight tendency to decrease by daily stripping (Table 1). However, the total number of spermatozoa expelled from each individual in Group c was usually higher than those from the other groups after second injection and mean was significantly higher (*P*<0.01). Total number of spermatozoa in the milt of some individuals from the Groups b and f were also higher than that of the control, but average numbers were not statistically different.

## Serum GtH

Serum GtH for each group is indicated in Fig. 2. In this experiment, although serum GtH level was measured 24 h after each injection, the GtH concentration was significantly higher in the group receiving 10 µg/kg of LHRH-a in NaCl. It is noteworthy that the GtH concentration of some individuals of this group became higher after 4th and 5th injections when the spermiation had been augmented. GtH levels in the group injected with 1 µg/kg in saline solution and with 10 µg/kg as well as with 1 µg/kg in propylene glycol were not different with the control.

In Group c, GtH concentration was decreased after final injection. However, even 3 days later, the levels of some individuals were still relatively
<figure><img src="image_4.png" /><figcaption>Fig. 3. Changes of serum 11-ketotestosterone (mean±SD) after injection of LHRH-a. Levels of some individuals in the group receiving 10 µg/kg b.w. of LHRH-a in 0.7% NaCl solution was increased. *; significant at *P*<0.05, **; *P*<0.01.</figcaption></figure>

high.

Two individuals which were injected with 10 µg/ kg of LHRH-a 2 days after the final injection expelled a large volume of milt and their GtH levels were elevated again (48.06 and 11.80 ng/ml, respectively).

## 11-Ketotestosterone

Serum level of 11-ketotestosterone was increased after injection of 10 µg/kg of LHRH-a in saline solution (Fig. 3). The elevated level was sustained during the whole period of injection, but 11-ketotestosterone decreased 2 days after final injection. LHRH-a at 1 µg/kg in saline solution or 10 µg/kg in propylene glycol evoked slight elevation of this steroid in the serum. However, these were not statistically significant.

Comparing with the changes of GtH level in the serum, it is obvious that 11-ketotestosterone has increased more rapidly and uniformly than GtH. Namely, the levels of the steroid were already high on the day after the first injection, while the concentration of the GtH was relatively low during the first 2 days.

The levels of the steroid in two individuals again receiving 10 µg/kg of LHRH-a 2 days after final injection were high; one was 32.78 ng/ml and the other 13.42 ng/ml.

## Discussion

In this study, the authors examined the effects of repeated administration of synthetic LHRH analogue to adult male carp. One of the results obtained was that the LHRH-a at the dose of 10 µg/kg in 0.7% NaCl was the most effective to augment milt collection. This result indicates that the LHRH-a is a highly effective agent for stimulating spermiation in the common carp.

Collectable amounts of milt and the total number of spermatozoa from the fish injected with 10 µg/ kg in 0.7% NaCl (Group c) were greater compared with sperm collections from fish receiving 1 µg/ kg in 0.7% NaCl or 10 µg/kg in propylene glycol. One µg/kg in propylene glycol was regarded as non-effective in this experiment. These results indicate that pure propylene glycol is not suitable as the vehicle for the LHRH-a. However, WELL and CRIM<sup>10)</sup> found that LHRH-a dissolved in 40% of propylene glycol had a lesser effect on spermiation of *Salmo salar* compared with the peptide dissolved in saline or in cholesterol pellets. Therefore, more effective vehicles to administrate LHRH-a must be considered in case of carp from practical point of view.

In this study, serum GtH was increased after
LHRH-a injection. The increment was more apparent in the group receiving 10 µg/kg in 0.7% NaCl. Similar augmentation of GtH in the male after LH-RH injection was also reported in several fishes.⁵,⁸⁻¹⁰) These results indicate that the discharge of GtH from the pituitary gland is evoked by a mammalian LHRH or analogue.

Serum 11-ketotestosterone was increased after LHRH-a injection in this study. Steroid hormones which might have influence on testicular activity, i. e., spermatogenesis, spermiogenesis and spermiation were already examined by several researchers. For instance, YAMAZAKI and DONALDSON²¹) indicate that the spermiation in goldfish was regulated by 17-methyltestosterone and dehydroepiandrosterone. It was found that concentration of 11-oxotestosterone was high in spermiating rainbow trout male²⁰, ²¹) while very low levels are present in females.²²) CAMPBELL et al.²²) proposed a hypothesis that this steroid was an important androgen in the regulation of testicular activity and spermiation. SCOTT et al.²³) also pointed out that the peak levels of 11-ketotestosterone coincided with the period of spermiation in rainbow trout. Furthermore, DÉPÈCHE et al.²⁴) mentioned that at final stage of spermatogenesis, most of the radioactivity was recovered in a derivative identified as 11-ketotestosteron when incubated the testis with the radioactive precursor.

On the other hand, HUNT et al.²⁵) reported that plasma 11-ketotestosterone in Salmo salar was even decreased at the time of spermiation. Moreover, UEDA et al.²⁶) reported that plasma 17α,20β-dihydroxyprogesterone levels increased and maintained during the period of active spermiation in precociously mature amago O. rhodurus rhodurus. They considered based on their result that the physiological action of the steroid may involve spermiation by augmentation physiological action of hydration. However, DÉPÈCHE et al.²⁴) considered that 17α,20β-dihydroxyprogesterone in rainbow trout male might act upon some step of the mitotic cycle and meiotic process of the male germ cells, because of high levels in the plasma during early reproductive cycle.

Although the concept of endocrinological control on the testicular activity especially spermiation has been somewhat controversial among species as described above, the results obtained in this experiment clearly demonstrated that the amount of milt and 11-ketotestosterone in the serum were both increased in connection with

augmentation of GtH after LHRH-a injection in carp. This result implies an important role of the steroid on the spermiation and sperm moving into the sperm duct. Mechanisms of endocrine control on this subject require further study. The role of other steroids also should be further analyzed.

The total number of the spermatozoa in the milt expelled from the males which were injected with 10 µg/kg LHRH-a in 0.7% NaCl was 10 folds greater than the control and the total amount of milt was increased more than 24.5 times. These results indicate that not only spermiation but also the production of seminal plasma was stimulated by LHRH-a administration. Under the influence of GtH seminal plasma is thought to be secreted from the wall of testicular lobules especially cyst cells.²⁷) In addition, the present authors (FT and RB) observed electronmicroscopically that salmon GtH stimulates the secretory response of epithelial cells of sperm duct as well as cyst cells in the testis in case of rainbow trout.*¹ This phenomenon shall be published in another paper.

We demonstrated in this study that milt collection in carp during spawning season was easily possible after administration of LHRH-a. Repeated sperm-taking is also facilitated in rainbow trout by LHRH-a or salmon GtH as well as pituitary powder.*² Improvement of this kind of technique should be expected to contribute to reducing the cost for seed production and to develop the hybridization.

This work was partly supported by a grant CNEXO-CNRS "les bases biologiques de l'aquaculture". This work was carried out in France and FT received a research grant from the Japanese Ministry of Education for complementary experiments.

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