Injection Of Carp Pituitary Extract Research Articles

Ovarian lavage / artificial insemination method as an alternative approach for controlled reproduction of the northern pike (Esox lucius)

This study aimed to compare the effectiveness of the ovarian lavage / artificial insemination method with the traditional hormonal administration and fertilization methods over the artificial reproduction of the northern pike (Esox lucius). For this purpose, groups of five females were treated as follows: intraperitoneal injection of saline (C1); ovarian lavage with saline (C2); intraperitoneal injection of carp pituitary extract (CPE, T1); ovarian lavage with CPE (T2); intraperitoneal injection of CPE and ovarian lavage with semen after 72 h (T3); ovarian lavage with CPE and ovarian lavage with semen after 72 h (T4). According to the results, no fish ovulated in the control groups (C1 and C2). There were no significant differences (n.s.) among experimental treatments (P > 0.05, n.s.) in the reproductive parameters, such as latency time, ovulation rate, stripped egg amount, and pseudo-gonadosomatic index. The lowest fertilization rate (54.8%) was observed in the T4 treatment and significantly differed from the T1 and T2 treatments (P < 0.05). Moreover, the highest survival at swim-up stage was measured in the T4 treatment with a significant difference compared to the T1 group (P < 0.05). The survival at the eyed-egg stage, hatching rate, and malformations were similar (P > 0.05, n.s.) in all applied strategies. The results demonstrated that the ovarian lavage / artificial insemination method could be applied to control northern pike reproduction, like the traditional fertilization method. Consequently, this novel technique can be suggested as an alternative strategy to facilitate the hatchery operations in the controlled reproduction of this species.

Induced spawning and embryonic and early larval development of bonefish (Albula vulpes).

Bonefish (Albula vulpes L.) are a highly prized sport fish. Despite their economic importance, populations in the Florida Keys and Caribbean are in decline, with the early life history undescribed. Injections of carp pituitary extract into A. vulpes during the advanced stages of ovarian development induced ovulation and spawning. Embryos were sampled hourly until hatching into undeveloped, yolk-sac leptocephalus larvae. These larvae survived 56 h post-hatch, when myomeres and eyes were developing but not the mouth. These results inform future research on the reproduction and early life history of A. vulpes.

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation

Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out.An experimental group of silver-stage eel females was maintained at 18°C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development.These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel ( Anguilla anguilla) females

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshβ and lhβ expression, plasma 17β-estradiol (E2) and vitellogenin, estrogen receptor 1 ( esr1), and vitellogenin 2 ( vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17 °C was compared with a constant 20 °C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshβ, lhβ, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshβ expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

Intergeneric Hybridization between Kutum, Rutilus frisii kutum, and Bream, Abramis brama orientalis, of the Caspian Sea

Abstract Artificial hybridization was performed between Rutilus frisii kutum and Abramis brama orientalis of the Caspian Sea. Synchronization of spawning of female broodstock of both species was induced by injection of carp pituitary extract. Reciprocal crossings between R. frisii kutum ♀×A. brama orientalis ♂ (RA) and A. brama orientalis ♀×R. frisii kutum ♂ (AR) produced viable hybrid larvae without any clear particular pre‐ or postzygotic isolation phenomena. RA and AR hybrid larvae were reared to fingerling stage with survival rates of 22.5 and 28% and average weight of 6.8 ± 0.17 g and 9.0 ± 0.79 g, respectively. A heterosis of 45% was calculated for weight at fingerling stage. RA and AR hybrid fingerlings were cultured in polyculture along with Chinese carps for 6–7 mo and reached an average weight of 190–195 g and 235–255 g, respectively. Karyotyping of these hybrids revealed a modal diploid number of 2n = 50 for both groups, which is similar to those of the parental species. Discriminant function analysis on 28 morphometric and meristic characteristics of two parental species as well as their hybrids could separate these groups at highly significant level (P &lt; 0.001). These results indicated an overall intermediate inheritance of the studied characters.

Release of sex steroids into the water by roach

Electro‐olfactogram (EOG) recordings of the olfactory epithelium of both male and female roach Rutilus rutilus demonstrated that both sexes were able to detect free and glucuronidated 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) with high sensitivity. Male, but not female, roach were also sensitive to androstenedione. Sexually mature female roach were shown to release free 17,20β‐P, glucuronidated 17,20β‐P and androstenedione into the water; for all three steroids, the rate of release was significantly enhanced by injection of carp pituitary extract (CPE). A series of trials was also carried out which showed that mature males, and to a lesser extent immature males and females, were able also to release free and glucuronidated 17,20β‐P, both before and after CPE treatment. Water extracts from containers that had held CPE‐treated mature male and female roach were examined for the presence of other steroids. This revealed that free and glucuronidated 17,20β‐P plus free and glucuronidated 17,20β,21‐trihydroxy‐4‐pregnen‐3‐one (17,20β, 21‐P) predominated in water extracts from both sexes. The free moieties of 17,20α‐dihydroxy‐4‐pregnen‐3‐one, 17‐hydroxyprogesterone and 11‐deoxycortisol were found at concentrations which were between four and 20 times lower than those of free 17,20β‐P. Androstenedione was found at concentrations which were 25‐fold lower than those of 17,20β‐P. Despite its apparent high rate of release by sexually mature male and female roach, free 17,20β,21‐P was found not to exhibit any EOG activity at the highest dose tested (10−7 M).

Channel catfish follicle-stimulating hormone and luteinizing hormone: complementary DNA cloning and expression during ovulation.

Gonadotropins are important regulators of reproduction. To develop molecular resources for production of recombinant gonadotropins, we have cloned and sequenced complementary DNA encoding the channel catfish (Ictalurus punctatus) follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which encode 132 and 140 amino acid proteins, respectively. The deduced amino acid sequences of the catfish FSH and LH are highly conserved with those cloned from other teleosts. Both the FSH and the LH were highly induced during ovulation after injection of carp pituitary extract. Taken together with our previous findings of enhanced expression of growth hormone and other pituitary hormones, this research suggests that a hormonal cocktail may be needed for more efficient manipulation of catfish reproduction. The availability of the catfish FSH and LH cDNAs provides the opportunity for production of immunologically or biologically active recombinant gonadotropins for the study of catfish reproductive physiology and the manipulation of artificial spawning for aquaculture.

Transcriptional activities in the pituitaries of channel catfish before and after induced ovulation by injection of carp pituitary extract as revealed by expressed sequence tag analysis.

Expressed sequence tag (EST) analysis was adopted to address physiological changes after injection of carp pituitary extract for induction of ovulation. ESTs were analyzed from cDNA libraries constructed from mRNA isolated from channel catfish (Ictalurus punctatus) pituitaries before and after induction of ovulation by injection of carp pituitary extract. One hundred randomly picked clones were analyzed. Of the sequences generated, a large percentage (59%) of ESTs were identified as known genes by identity comparisons. These 59 clones of known gene products represent transcriptional products of 30 genes. The 41 clones of unknown gene products represent 33 genes. Expression of gonadotropin (GtH) alpha-subunit (149%) and prolactin (176%) was slightly enhanced as a result of induced ovulation. Large increases in frequencies of several peptide hormones were observed as a result of induced ovulation: GtH beta-I, 486%; GtH beta-II, 933%; growth hormone, 393%; proopiomelanocortin (POMC), 345%. POMC represented about 21% of all transcriptional activity in the pituitaries after induced ovulation. This is the first study addressing physiological changes after injection of carp pituitary extract, a procedure widely used in catfish hatcheries.

Artificial induction of gonadal maturation and ovulation in the Japanese eel (Anguilla japonica T. et S.)

injections of carp pituitary extract (CPE, 5 pit. / fish / 10 days) induced ovarian maturation in female eel, gonadosomatic index (GSI) increasing significantly to 43-55 %. 4 injections of CPE (1-2 pit. / fish / 10 days) induced spermatogenesis and spermiation in male eel, GSI increasing to 2.1-2.8 %. After serial injections of CPE, serum gonadotropin (GtH) levels increased significantly, indicating that the exogeneous GtH stimulated gonadal development and maturation in the Japanese eel. 8 implantations of 17 a-methyltesto sterone (MT) or androstenedio ne (ADSD) (50 ug / g body weight /15 days) stimulated ovarian maturation in female eel, GSI increasing significantly to 38-49 %. 4 implantations of MT or ADSD (50 ug / g body weight /15 days) induced spermatogenesis and spermiation in the male eel, GSI increasing to 2.1-3.5 %. MT or ADSD implantations significantly elevated pituitary and serum GtH contents. ADSD or MT implantations resulted in the significant elevation of serum estradiol levels in female eel and serum testosterone levels in male eels. In female eel, ADSD implantations significantly increased mammalean-libe-g onadotropin-rele asing hormone (mGnRH) content in brain and pituitary. These results demonstrated that the positive feedback effects of sex steroids on the brain (hypothalamus)-p ituitary-gonad axis play an important role on the induction of gonadal development and maturation in the Japanese eel, and the endogeneous GtH is the key hormone during these physiological processes. In the matured female eel, ovulation and spawning can be induced 18-21 hours following an injection of CPE alone or in combination with (D-Ala 6, Pro 9 -NEt)-lutein izing hormone-releasin g hormone (LHRH-A) and dopamine antagonist domperidone (DOM).

Monthly variation in sperm motility in common carp assessed using computer‐assisted sperm analysis (CASA)

Sperm motility variables from the milt of the common carp Cyprinus carpio were assessed using a computer‐assisted sperm analysis (CASA) system across several months (March‐August 1992) known to encompass the natural spawning period. Two‐year‐old pond‐raised males obtained each month were primed with an exogeneous hormone injection of carp pituitary extract and stripped of milt 18–24h later. The milt was diluted, activated and videotaped using a high‐speed (200 Hz) videocamera and recorder. Videotaped samples were subsequently analysed using the CellTrak/S CASA system (Motion Analysis Corp.) for percent motility, curvilinear and straight‐line velocity. In addition to assessing changes in motility parameters across several months, a comparison was made between two predilution/ activation media combinations (homologous seminal plasma/NaCl+HEPES v. 2‐h incubation in 200 mM KCl+Tris/Tris). The percentage of motile cells assessed immediately after activation was significantly greater in the summer months (May, June, July) when compared to a spring sampling point (March); when assessed 1 min after activation, cells prediluted in seminal plasma maintained a higher percentage of motility than those prediluted in KC1. Curvilinear and straight‐line velocities exhibited a slight seasonal trend; variations in response to the predilution treatments were observed with these measurements. Sperm count gradually increased through April and May (9–63 × 109 to 2–38 × lO10ml– 1 milt), declined in June and July (to 1–83 × lO10ml– 1 milt), and was followed by a steep increase in August (2–74 × 1010 ml– 1 milt). Mean seminal plasma osmolality remained relatively constant (250–265 mOsmol kg – 1) throughout the sampling period.

Spermiation of common tench ( Tinca tinca L.) stimulated with injection or implantation of GnRH analogues and injection of carp pituitary extract

Spermiation was stimulated in the common tench, Tinca tinca L., by injection of carp pituitary extract (CPE; 0.5 mg · kg −1 body weight), carp homogenized pituitary (1 mg · kg −1 body weight), [ d-Arg 6,Pro 9-NHEt] salmon GnRH (20 μg · kg −1 body weight) and [ d-Ala 6,Pro-NHEt] mammalian GnRH slow-releasing implants at a dose of 25 μg per male (average, 652 g of body weight). The sperm was collected daily for 5 days post-stimulation and diluted with immobilizing solution (180 mM NaCl, 20 mM Tris, pH 7). Sperm density, expressed as billions per kg of body weight, was 47.31 for CPE, 49.42 for carp homogenized pituitary, 51.84 for ( d-Arg 6,Pro 9-NHEt] salmon GnRH, 28.93 for GnRH analogue implant and 13.73 for the control group treated with Ringer solution. The total number of spermatozoa obtained by stimulation with [ d-Arg 6,Pro 9-NHEt] salmon GnRH was significantly higher ( P < 0.05) than for the carp-hypophysis-treated groups. The best spermiation with the GnRH analogue implantant group was at 4 days after implantation ( P < 0.001).

Chapter 9 Isolation of Fish Sperm Flagella

Publisher Summary This chapter describes methods for purifying axonemes and the focus is on preparing intact flagella to analyze their membrane proteins and reactivate them to study axonemal motility mechanisms. Fish spermatozoa offer a good source from which to isolate and purify large amounts of vertebrate flagella and axonemes. Sperm from two teleost species, trout and carp, have been used extensively to study the mechanisms involved in the initiation of sperm motility. During the spawning season, an adult male trout produces a large number of sperm. In carp, sperm production can be induced throughout the year by repetitive injection of pituitary extracts. Trout and carp spermatozoa are quite simple and their flagella contain a classical 9+2 axoneme without periaxonemal structures. The absence of an acrosomal vesicle decreases the risk of contamination of the preparation with proteases. Trout sperm motility can be inhibited by media containing large amounts of potassium or having a low Ph. Carp sperm activation is controlled by a decrease in the external osmotic pressure and is not dependent on the external ions. It is advisable to include different protease inhibitors in extraction buffer (IMEN), as proteasomes are present on trout sperm plasma membrane. For trout, the water should be aerated with bubbling and frequently renewed. Chlorine must be avoided. For injection or sperm collection, fish must be anesthetized. For carp, spermiation can be induced throughout the year by intraabdominal injection of carp pituitary extract. Only a fraction of the sperm obtained from a single trout can be used directly for axoneme isolation. Therefore, for biochemical studies, some of the sperm may be frozen in liquid nitrogen for later use. The chapter discusses isolation of axonemes and flagella, and thereafter the demembranation and ATP reactivation of isolated carp sperm flagella.

Spermiation and sperm quality of European catfish (Silurus glanis L.) after implantation of GnRH analogues and injection of carp pituitary extract

Summary Spermiation was stimulated in the European catfish Silurus glanis L. by injection of carp pituitary preparation (5 mg kg-1 of body weight) and mammalian GnRH [(D-Ala6) en-RH ProNHEt] implanted at a dose of 75 μg and 150 μg per male, respectively. The total number of spermatozoa collected by daily sampling during 7 days post-stimulation and expressed in millions per kg of body weight was 3.29 with carp pituitaries, 0.872 and 0.780 with 75 μg and 150 μg GnRH anjoue implant, respectively, and 0.387 in the control group treated with Ringer solution. In all groups, after sperm stripping there was a spontaneous activation by urine of the sermatozoa motility which was revented by collection of the sperm in an immobilizing solution (NaCl 200 mM, Tris 30 mM, pH 7). The percentage of motile spermatozoa ranged from 80 to 65% at days 1 to 5, and less than 50% at day 6 and 7. A similar decline of quality was observed with time for mass progressive movement (55 to 65 s on day 1, and 28 to 35 s on day 7) and total duration of displacement (66 to 77 s on day 1, and 45 to 53 on day 7).

Effects of hormonal treatments on induced ovulation in gudgeon, Gobio gobio L.

The effectiveness was studied of different hormonal treatments to induce gamete emission in a wild stock of a European baitfish, the gudgeon ( Gobio gobio L.). Ovulation was induced by injection of a hypothalamic factor, the luteinizing hormone-releasing hormone superactive analog (D-Trp 6, des Gly 10-LHRH), in combination with pimozide (PIM), a dopamine antagonist, or by injection of a carp pituitary extract (CPE). More than 60% of treated females ovulated after injection of CPE (10.0 μg/g) and nearly 100% after injection of PIM+LHRH-A. There was a large variability in the time between injection and stripping and in fertilization success. Hatching rate varied between 0 and 95% and there was no relationship with latency time or ovulation index. Different techniques are proposed to resolve these problems.

Effects of pimozide and LHRH-Aa on carp (Cyprinus carpio L.) oocyte maturation and ovulationin vivo

Effects of pimozide (Pim) and [(D-Ala(6), Pro(9)-NEt) LHRH] (LRH-Aa) on common carp oocytes maturation and ovulationin vivo under laboratory and commercial fisheries farm conditions were investigated.Although injections of Pim and LRH-Aa at the doses of 10 mg and 50 µg/kg body weight respectively, did not increase mGtH levels (66.7-155.8 mg/ml) as much as injections of carp pituitary extract (chh) (382.1 ng/ml), induced GtH levels were high enough to induce ovulation. Changes in the ovary caused by Pim and LRH-Aa were similar to those induced by chh, and Pim injected together with LRH-Aa in a single injection gave the same results concerning ovulation induction as when they were applied separately at 6h interval.

Induction of ovulation in the loach ( Paramisgurnus dabryanus) using pimozide and [D-Ala 6, Pro 9-N-ethylamide]-LHRH

The effects of intraperitoneal injections of [D-Ala 6, Pro 9-N-ethylaminde]-luteinizing hormone releasing hormone (LHRH-A) and pimozide (PIM), a dopamine receptor antagonist, on ovulation in loach were investigated. LHRH-A and PIM administered separately were ineffective in inducing ovulation. However, injections of PIM and LHRH-A simultaneously or the injection of PIM 2.5–3 h prior to LHRH-A were highly effective means of inducing ovulation. The simultaneous injection of PIM (1.0 μg/g body wt) and LHRH-A (0.05 μg/g body wt) resulted in a greater ovulatory response than injection of carp pituitary extract (1 pituitary/fish).

The twenty-four-hour seminal hydration response in goldfish ( Carassius auratus)—I. sodium, potassium, calcium, magnesium, chloride and osmolality of serum and seminal fluid

1. 1. Spermiation was induced in goldfish by an intraperitoneal injection of carp pituitary extract. The composition of the serum and seminal fluid was studied 24 hr after the injection. 2. 2. The semen volume was greater and the water percentage of the testes was 7·1±0·6(S.E.) higher in the extract-injected fish than in the controls. 3. 3. The osmolality and Na, K, Cl, Mg and Ca levels in the seminal fluid did not change in spermiation, and the seminal fluid was isosmotic to the serum. 4. 4. An increase in Na and K in the whole testes was greater than that accounted for by the addition to the seminal fluid. 5. 5. Serum Na increased 3·4±1·2 m-equiv./1; serum K decreased 0·48 ± 0·19 m-equiv./1; total serum protein decreased 0·56±0·12 g/100 ml. Theosmalality and Cl, Mg and Ca level in serum did not change.