Effects of continuous administration of human chorionic gonadotropin, salmonpituitary extract, and gonadotropin-releasing hormone using osmotic pumps oninduction of sexual maturation in male Japanese eel, Anguilla japonica

Hirohiko Kagawa a,, Yosuke Kasuga a, Junichi Adachi b, Akifumi Nishi b, Hiroshi Hashimoto b,Hitoshi Imaizumi b, Shunji Kaji ba Department of Biological Production and Environmental Science, University of Miyazaki, Miyazaki 889-2192, Japanb Shibushi Station, National Center for Stock Enhancement, Fisheries Research Agency, Shibushi 899-7101, Japan

a b s t r a c ta r t i c l e i n f o

Article history:Received 7 April 2009Received in revised form 21 July 2009Accepted 22 July 2009

Keywords:Japanese male eelSexual maturationHuman chorionic gonadotropinSalmon pituitary extractGnRHOsmotic pump

The purpose of this study was to examine the effects of continuous administration of human chorionicgonadotropin (HCG), salmon pituitary extract (SPE), and gonadotropin-releasing hormone analogue(GnRHa) via an osmotic pump with a long-term sustained hormone-release system on the induction ofsexual maturation in male Japanese eels (Anguilla japonica). Implantation of a single HCG-loaded osmoticpump induced spermatogenesis and increased the gonadosomatic index (GSI) values at 3542 dayspostimplantation; GSI values were higher (10.71.0) than those of the fish implanted with a cholesterolpellet containing HCG (2.40.6). Implantation of an SPE-loaded osmotic pump also stimulatedspermatogenesis, although the GSI values were lower than those of the fish implanted with the HCG-loaded osmotic pump. Implantation of a single osmotic pump loaded with various doses of GnRHa (0.94,1.86, or 3.75 g/day) did not stimulate spermatogenesis. The GSI values and milt weight of the HCG-administered eels significantly increased at a dosage of 5 IU/day in a dose-dependent manner up to 50 IU/day. Sperm motility was not significantly different among the eels treated with different doses of HCG.Therefore, implantation of a single osmotic pump loaded with HCG (50 IU/day) is a reliable method forinducing spermatogenesis and spermiation in sexually immature Japanese eels.

2009 Elsevier B.V. All rights reserved.

1. Introduction

Cultivated male eels are sexually immature in general and arenever mature under commercial rearing conditions (Yamamoto et al.,1972). One exceptional report describes spermatogenesis and sper-miation of two cultivated male eels which were maintained undernatural environmental conditions (Matsubara et al., 2008). It has beenobserved that repeated injections of salmon pituitary extract (SPE)induce vitellogenesis in female eels (Kagawa, 2003; Kagawa et al.,2005; Ohta et al., 1996a; Yamamoto and Yamauchi, 1974; Yamamotoet al., 1974). Further, human chorionic gonadotropin (HCG) inducesspermatogenesis and spermiation in male eels (Ohta et al., 1996b):Repeated injections of HCG at 250 IU/fish (1 IU/g body weight) over10 weeks efficiently induced spermatogenesis and spermiation, and12 g of approximately 70% motile spermatozoa was obtained.However, weekly injection of HCG requires repetitive handling ofthe broodstock and substantial labor, time, and monitoring, resultingin stress to and increased mortality of the fish. Miura et al. (1991)

reported that a single injection of HCG can induce the completeprocess of spermatogenesis from the proliferation of spermatogonia tospermiogenesis. However, themaleswhich are induced tomaturity bya single injection of HCG produced little milt.

In recent studies on drug delivery systems in fish, a variety ofgonadotropin-releasing hormone analogue (GnRHa) delivery systemshave been developed for sustained hormonal release. These includecholesterol pellets (Weil and Crim, 1983; Zohar and Mylonas, 2001);microspheres prepared using copolymers of lactic and glycolic acids(LGA; Mylonas et al., 1993; Zohar et al., 1995) and nondegradablecopolymers of ethylene and vinyl acetate (EVAc; Mylonas et al., 1998;Zohar, 1996); as well as other copolymer pellets (Hirose et al., 1990;Matsuyama et al., 1993). These hormone delivery systems effectivelyinduce oocyte maturation and ovulation or spermiation in reproduc-tively dysfunctional cultured fish (Zohar and Mylonas, 2001).However, no reports have investigated the effects of long-term(N1 month) sustained-release delivery systems of gonadotropins onsexual maturation in such fish.

An osmotic pump is a delivery device for long-term administrationof drugs and hormones. Implantation of a single osmotic pump loadedwith a gonadotropic hormone (e.g., pregnant mare serum gonado-tropin or HCG) has been carried out experimentally in mammals

Aquaculture 296 (2009) 117122

Corresponding author. Tel./fax:+81 985 58 7223.E-mail address: kagawa@cc.miyazaki-u.ac.jp (H. Kagawa).

0044-8486/$ see front matter 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2009.07.023

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Racquel IsadaRacquel IsadaRacquel IsadaRacquel IsadaRacquel Isadamailto:kagawa@cc.miyazaki-u.ac.jphttp://dx.doi.org/10.1016/j.aquaculture.2009.07.023http://www.sciencedirect.com/science/journal/00448486

(Gibson et al., 1994; Patton et al., 1990) and birds (Girling et al., 2002;Wakabayashi et al., 1996). However, the use and efficacy of osmoticpumps for inducing sexual maturation have not been studied inteleosts. Therefore, in this study, we examined the effects of HCG, SPE,and GnRHa administration via an osmotic pump which has a long-term sustained hormone release on the induction of sexualmaturationin male Japanese eel Anguilla japonica.

2. Materials and methods

2.1. Fish and hormonal treatments

Culturedmale Japanese eelswere obtained from a fish farm or fromthe Shibushi Station, National Center for Stock Enhancement, FisheriesResearch Agency, Japan. After acclimation to seawater, they weremaintained without food in indoor 400-L circulating tanks under anatural photoperiod at a water temperature of 20 C. After anesthe-tizationwith 2-phenoxyethanol (Nacali Tesque, Tokyo, Japan), the fishwere weighed and then treated with the various hormones.

A newly introduced osmotic pump (Osmotic Pump Type 2002; AlzetOsmotic Pumps Co., Cupertino, CA; diameter=7 mm, length=30 mm,reservoir volume=approximately 200 l) that can release constantamounts of hormones for a long period was loaded with variousamounts of GnRHa, HCG, or SPE. According to the manufacturer'sinstructionmanual, this osmotic pump can release 5 l of a solution perday for approximately 4550 days when the fish are maintained at awater temperature of 20 C.We confirmed that the expected volume ofhormone solutions is retained in the osmotic pump reservoir at the endof experiment.Moreover, constantHCGrelease fromtheosmotic pumpswas confirmed by measuring serum levels of HCG during eachexperiment (data not shown). An osmotic pump was implanted intothe peritoneal cavity of eacheel after cutting the abdomenwith a scalpelapproximately 8 mm. The wound was not sutured, but it was healed in2 weeks.

HCG (Teikoku Zhoki Co. Ltd., Tokyo, Japan) was dissolved in 0.9%sterilized sodium chloride solution. GnRHa, des-Gly10-[D-Ala6]-LH-RHethylamide (Sigma), was dissolved in 0.9% sodium chloride contain-ing 0.1% bovine serum albumin (BSA), which was added to preventthe adhesion of GnRHa onto the reservoir wall of the osmotic pump.SPE was prepared by homogenizing salmon (Oncorhynchus keta)pituitary powder with 0.9% sodium chloride solution, followed bycentrifugation at 9700g (Kagawa et al., 1995, 1997, 1998). The SPEwas concentrated with a concentrator (Vivapore 10/20; SartoriusStedim Lab, Ltd., Gloucestershire, UK) before loading into the osmoticpumps at a concentration of 22.5, 45, or 90 mg/200 l. Cholesterolpellets were prepared according to the method applied in ourprevious study (Kumakura et al., 2003; Matsuyama et al., 1992). Thepellets (diameter=2 mm, length=6 mm) containing HCG (1500 IU/pellet) were stored at 30 C until use. Each cholesterol pellet wasimplanted into the peritoneal cavity by using a 2-mm trocar.

In experiment 1, twenty-four fish (mean body weight=314.03.1 g, range=281.0343.0 g) were randomly divided into four groups(n=6 eels/group): the saline control group, the cholesterol pelletgroup, the osmotic pump group, and the injection group. HCGwas administered by injection (330 IU/week), cholesterol pellets(1500 IU/pellet), and osmotic pump (50 IU/day). The injection groupreceived intraperitoneal injections of HCG once a week (5 timestotally). The fish in the cholesterol pellet group and osmotic pumpgroup received a single pellet and a single osmotic pump, respectively,on the first experimental day.

In experiment 2, thirty fish (mean body weight=232.73.9 g,range=201.3277.7 g) were randomly divided into six groups (n=5eels/group). The eels were implanted with a single osmotic pumploadedwith various doses of SPE (0.56, 1.12, or 2.24 mg/day) or with asingle osmotic pump loaded with HCG (50 IU/day). The eels wereimplanted with a single osmotic pump loaded with various doses of

SPE (0.56, 1.12, or 2.24 mg/day). The saline control group received asingle osmotic pump containing 0.9% sodium chloride. The initialcontrol fish were sampled on the first experimental day.

In experiment 3, thirty-five fish (mean body weight=234.89.2 g,range=171.3367.2 g) were randomly divided into seven groups(n=5 eels/group). The eels were implanted with a single osmoticpump loaded with various doses of GnRHa (0.9, 1.8, or 3.6 g/day), asingle osmotic pump loaded with HCG (50 IU/day), or a single osmoticpump loadedwith SPE (2.24 mg/day). The saline control group receiveda single osmotic pump containing 0.9% sodium chloride. The initialcontrol fish were sampled on the first experimental day.

In experiment 4, forty-eight fish (mean body weight=336.710.3 g, range=253.0532.0 g) were randomly divided into six groups(n=8 eels/group). The eels were implanted with a single osmoticpump loaded with various doses of HCG (1, 5, 10, 25, or 50 IU/day).The saline control group received a single osmotic pump containing0.9% sodium chloride.

2.2. Sampling

At 35 days postimplantation in experiment 1, and at 42 dayspostimplantation in experiments 2, 3, and 4, the eels were terminallyanesthetized with 2-phenoxyethanol and weighed. Their milt andgonads were sampled, and the gonadosomatic index (GSI), milt weight,and sperm motility were calculated. Survival rates of experimental fishin each experimentwere 75% (experiment1), 90% (experiment 2), 100%(experiment 3), and 98% (experiment 4), respectively.

2.3. Measurement of milt weight and spermatocrit

The milt weight and spermatocrit were measured according to aprevious report (Ohta et al., 1996b). Briefly, the area around the genitalpore was rinsed with distilled water, dried with a soft towel, and miltwas collected by using a preweighed Pasteur pipette and small tube.Care was taken to avoid contamination of the milt with seawater orurine. The milt from the treated fish was obtained by application ofgentle pressure on the abdomen. The milt weight was calculated fromthe difference in weight of the Pasteur pipette and small tube beforeand after measurement. Spermatocrit ([packed cell volume/total miltvolume]100) values were determined for each milt sample bycentrifuging the capillary tubes (12000 rpm for 15 min).

2.4. Sperm motility

The milt was maintained on crushed ice in a cool box until theassessment of sperm motility. According to the method used in aprevious study (Ohta et al., 1996b), 1-l milt was collected with amicropipette and diluted in 1 ml 450-mM sodium chloride bufferedwith 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES) and sodium hydroxide (pH 7.6) in 24-well culture plates(Corning Inc., Corning, NY). Immediately after agitation of the dilutedmilt ~20 l of the mixture was mounted onto a glass slide with amicropipette and sperm motility was observed under a lightmicroscope. As reported in a previous study (Viveiros et al., 2003),spermmotility was subjectively classified according to the percentageof motile spermatozoa showing forward movement as follows: 0, nomovement; 1, up to 25% of the cells are motile; 2, up to 50% of the cellsaremotile; 3, up to 75% of the cells are motile; and 4, more than 75% ofthe cells are motile. Measurement of each sample was repeated thriceby three persons and the data were averaged.

2.5. Histology

Parts of the testes obtained at the end of the experiments werefixed in Bouin's solution. Serial 6-m-thick paraffin sections werestained with Mayer's hematoxylin and eosin.

118 H. Kagawa et al. / Aquaculture 296 (2009) 117122

2.6. Statistics

All data are represented as the meanSEM. The differences werecompared by one-way ANOVA and the NewmanKeuls multiplecomparison test. A result of pb0.05 was considered statisticallysignificant. The fish that died during the experiment were omittedfrom the evaluation.

3. Results

3.1. Experiment 1

In the control group, the mean GSI was below 0.2 (Fig. 1A) and thefish had very small immature testes (Fig. 2A) in which onlyspermatogonia was observed, and no developing germ cells, such asspermatocytes, spermatids, and spermatozoa (Fig. 3A). The implanta-tion of a single HCG-loaded osmotic pump significantly increased theGSI value (10.71.0) of the male eels (Fig. 1A), which had well-developed testis (Fig. 2B) with germ cells at various developmentalstages (Fig. 3B); the values were similar to that of the injection group(9.51.5). The mean GSI value of the cholesterol pellet group (2.40.6) also significantly increased when compared with the controlgroup, although the levels were much lower than those of the osmoticpump group (Fig. 1A). Histological observation revealed that variousdevelopmental stages of germ cells (from spermatogonia to sperma-tozoa) were present in the testes (data not shown). Milt weight washighly variable in the osmotic pump group and as a consequence therewere no significant differences between the osmotic pump group andthe injection group (Fig. 1B). Very small amounts of milt (b0.01 g)were obtained in the cholesterol pellet group.

3.2. Experiment 2

In both the initial control and saline control groups, the mean GSIvalue was below 0.2 (Fig. 4), and the fish had very small immaturetestes, as in the control fish of experiment 1. Implantation of anosmotic pump loadedwith various doses of SPE tended to increase theGSI, although no statistically significant differences were observed.

Fig. 1. Effects of human chorionic gonadotropin (HCG) on the gonadosomatic index(GSI, A) and the milt weight (B) of male Japanese eels, administrated via cholesterolpellet (CH, 1500 IU/pellet), osmotic pump (OS, 50 IU/day), or weekly injection(Injection, 330 IU/injection/week). The number in parentheses indicates the numberof fish used in each treatment. The letters indicate statistically significant differences(Pb0.05).

Fig. 2. Photographs of (A) the testis of a male Japanese eel implanted with a singleosmotic pump loaded with 0.9% sodium chloride (control) and (B) that of a maleJapanese eel implanted with a single osmotic pump loaded with human chorionicgonadotropin (HCG, 50 IU/day). The arrows indicate the testes and the arrowheadindicates the osmotic pump.

Fig. 3.Microphotographs of (A) the testis of a male Japanese eel implanted with a singleosmotic pump loaded with 0.9% sodium chloride (control) and (B) that of a maleJapanese eel implanted with a single osmotic pump loaded with human chorionicgonadotropin (HCG, 50 IU/day). Spermatogonia (SG) dominantly occupy the testis ofthe control fish. Germ cells at various developmental stages (SC, spermatocyte; SZ,spermatozoa) can be observed in the testis of the HCG-administered eel. Scalebar=100 m.

119H. Kagawa et al. / Aquaculture 296 (2009) 117122

Histological observations showed that the administration of SPE atthe 2.24 mg/day dose stimulated spermatogenesis (data not shown).Implantation of the HCG-loaded osmotic pump significantly increasedthe GSI value (1.90.6) when compared with that of the controlgroup.

3.3. Experiment 3

In both the initial control and saline control groups, the mean GSIvalue was below 0.15 (Fig. 5), and the fish had very small immaturetestes. No significant increase in the GSI value was observed in theGnRHa-administered groups when compared with the control group.Histological observations showed that only spermatogonia werepresent in the testes, as in the initial control group (data notshown). The SPE-loaded osmotic pump significantly increased the GSIvalue (3.81.2) when compared with that of the control fish (0.20.02). Implantation of the HCG-loaded osmotic pump also signifi-cantly increased the GSI value (8.32.6) when compared with that ofthe control group.

3.4. Experiment 4

Implantation of an HCG-loaded osmotic pump significantlyincreased the mean GSI value in a dose-dependent manner(Fig. 6A). GSI values significantly increased in the fish administered5, 10, and 25 IU/day of HCG and reached the maximum level at the50 IU/day dose.

Spermiation was observed in all the HCG-administered experi-mental groups, but milt volume in the fish administered the 1 IU/daydose was insufficient to be weighed by means of the method used inthis study (Fig. 6B). The highest amount (1.320.30 g) was obtainedfrom the fish administered with the 50 IU/day dose. Spermiation wasnot observed in the control group.

Sperm motilities of the groups administered 5, 10, 25, and 50 IU/day of HCG were 1.10.4, 1.10.3, 1.60.4, and 1. 40.3,respectively (Fig. 6C). Significant differences were not observedamong the experimental groups. Spermatocrit values were high in allthe treatment groups, and no significant differences were observedamong them (Fig. 6D).

4. Discussion

This is the first study demonstrating that implantation of a singleosmotic pump loaded with HCG stimulates spermatogenesis andspermiation in sexually immature male Japanese eels. The GSI valuesof the osmotic pump group were similar to those of the injection

group. Therefore, implantation of a single HCG-loaded osmotic pumphas the same potency as repeated injections to induce sexualmaturation in these male eels. Our data are comparable with thoseof the previous study showing that repeated weekly injection of HCG(~1 IU/g body weight) in immature male Japanese eels inducesspermiation after the sixth injection (Ohta et al., 1996b).

Implantation of the cholesterol pellet containing HCG alsostimulated spermatogenesis and spermiation, but the GSI values andmilt weight were much lower than those of the male eels implantedwith the osmotic pump. Cholesterol pellets have been used as asustained-release delivery system for GnRH mainly to induce oocytematuration and ovulation in many commercially viable fish species(Zohar and Mylonas, 2001). Moreover, our previous study showedthat implantation of cholesterol pellets containing GnRHa intoprepubertal red sea bream induces sexual maturation and ovulation(Kumakura et al., 2003;Matsuyama et al., 1995). Although the reasonsfor the lack of induction of complete sexual maturation on implanta-tion of the cholesterol pellets could not be clarified, it is possible thatthe cholesterol pellets did not release sufficient amounts of HCG toinduce sexual maturation in the eels. As cholesterol pellets have beendeveloped to deliver GnRHa for a relatively long period (Mylonas andZohar, 2001), they are adequate for release of GnRHa, but a proteinhormone, such as HCG, may not be released constantly for a long time.Moreover, release rates of GnRH from cholesterol pellets are notconstant; releasing rates are high during the first few days anddecreased to low levels in 10 days (Matsuyama et al., 1995).Therefore, an osmotic pump is a reliable device to deliver hormones,especially protein hormones, continuously for a prolonged period.

Implantation of a single osmotic pump loaded with SPE at variousdoses did not increase the GSI values of the eels in experiment 2. TheGSI values were very low and not significantly different from those ofthe control group. However, histological analysis showed that SPEstimulated spermatogenesis at least at the 2.25 mg/day dose. More-over, in experiment 3, implantation of an osmotic pump loaded withthe same amount of SPE as in experiment 1 significantly increased theGSI values in the eels. These are the first findings indicating that SPEcan stimulate spermatogenesis in male eels. As previously reported,SPE is used commonly for inducing sexual maturation in femaleJapanese eels (Kagawa et al., 2003; Yamamoto et al., 1974), but hasnot been used to induce sexual maturation in male eels. Therefore,these data indicate that SPE can stimulate both spermatogenesis andoogenesis in Japanese eels. However, the GSI values of the SPE-administered eels were lower than those of the HCG-administeredeels in experiments 1 and 2, suggesting that HCG has a higher potencythan SPE for inducing spermatogenesis at least at the doses used in

Fig. 4. Effects of a single implantation of osmotic pumps loaded with various doses ofsalmon pituitary extract (SPE, 0.56, 1.12, or 2.24 mg/day) or human chorionicgonadotropin (HCG, 50 IU/day) on the gonadosomatic index (GSI) of male Japaneseeels. The control fish (Control) were implanted with a single osmotic pump loaded with0.9% sodium chloride. Male eels were sampled as the initial controls (Initial control) atthe beginning of the experiment. The number in parentheses indicates the number offish used in each treatment. The letters indicate statistically significant differences(Pb0.05).

Fig. 5. Effects of a single implantation of osmotic pumps loaded with various doses ofgonadotropin-releasing hormone analogue (GnRHa, 0.9, 1.8, or 3.6 g/day), humanchorionic gonadotropin (HCG, 50 IU/day), or salmon pituitary extract (SPE, 2.24 mg/day) on the gonadosomatic index (GSI) of male Japanese eels. The control fish (Control)were implanted with a single osmotic pump loaded with 0.1% bovine serum albumin(BSA) in 0.9% sodium chloride. Male eels were sampled as the initial controls (Initialcontrol) at the beginning of the experiment. The number in parentheses indicates thenumber of fish used in each treatment. The letters indicate statistically significantdifferences (Pb0.05).

120 H. Kagawa et al. / Aquaculture 296 (2009) 117122

this study. The differences in the GSI values in the male eels treated ina similar manner (implantation of a single osmotic pump loaded withSPE or HCG) in experiments 1 and 3 may be influenced by thedifferences in the gonadal developmental stages at the initial stage(Miura et al., 1997) or by the individual differences in the HCGclearance rates of the fish (Ohta and Tanaka, 1997).

Implantation of a single osmotic pump loaded with GnRHa did notstimulate spermatogenesis in themale eels at the concentrations used.The GSI values and histological observations did not show anysignificant difference from those of the control group. GnRH treatmentalone (Dufour et al., 1991) of sexually immature female silver eels or incombination with testosterone to sexually immature female stripedbass (Holland et al., 1998) has often been unsuccessful in inducingvitellogenesis. The previous studies indicate that GnRH alone does notstimulate luteinizing hormone synthesis and release, resulting in afailure to induce sexual maturation (Dufour et al., 1991; Yaron et al.,1995). Combined treatments with GnRHa and a dopamine antagonist(blocker of dopamine D2-type receptor or blocker of catecholaminesynthesis) trigger luteinizing hormone release and vitellogenesis infemale European eels pretreated with estradiol-17 or testosterone(Dufour et al., 1988, 2003), although the oocytes attain only theprimary yolk stage and never develop to more advanced stages.Therefore, it is possible that an osmotic pump loaded with bothGnRHa and a dopamine antagonist may progressively induce sper-matogenesis in male Japanese eels.

The present study has shown that long-term treatment with HCG-administered via an osmotic pump is an effective method for inducingspermatogenesis and spermiation in male Japanese eels. The GSIvalues and milt weight of the eels administered HCG significantlyincreased at a dose of 5 IU/day in a dose-dependent manner up to50 IU/day. Our recent data also indicated that GSI, milt weight andsperm motility did not significantly increase at higher doses of HCG(75 IU/day and 100 IU/day). Combinedwith the present data on spermmotility, it can be concluded that a dose of 50 IU/day is the mosteffective dose for inducing spermatogenesis and spermiation in maleJapanese eels. The effective dose (50 IU/day) demonstrated in thisstudy is approximately the same as the previously reported effectivedose for weekly injections (1 IU/g body weight/week; Ohta et al.,

1996b). The milt weight (~1.3 g) and spermmotility (~20%) obtainedfrom the male eels at 42 days (6 weeks) after HCG administration(50 IU/day) were relatively lower than those reported in previousstudies (Ohta et al., 1996b). This variation may be due to the differentday of milt weight and sperm motility measurement. In the previousstudy, the samples were obtained after more than 10 weeks ofinjection, indicating that themilt weight and spermmotility graduallyincrease from 6 weeks onward and reach the maximal levels after10 weeks, following which they remain constant. Therefore, if HCG isadministered via an osmotic pump for more than 10 weeks, a usefulquantity and good quality milt might be obtained. Recent studies inour laboratory have demonstrated that approximately 1 g of sperma-tozoa with relatively high sperm motility (4080%) can be obtainedfrom male Japanese eels receiving prolonged HCG treatment(N10 weeks) via an osmotic pump (Kagawa et al., in preparation).

Acknowledgements

This work was supported by Grants-in-Aid from the Agriculture,Forestry, and Fisheries Research Council to H. Kagawa.

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Effects of continuous administration of human chorionic gonadotropin, salmon pituitary extract,.....IntroductionMaterials and methodsFish and hormonal treatmentsSamplingMeasurement of milt weight and spermatocritSperm motilityHistologyStatisticsResultsExperiment 1Experiment 2Experiment 3Experiment 4DiscussionAcknowledgementsReferences