Adult Male Monkeys Research Articles

Effect of α-Chlorohydrin on the Male Reproductive Organs of the Indian Langur: (Presbytis entellus entellus Dufresne)

A subtoxic dose of alpha-chlorohydrin (3-chloro-1, 2-propanediol, U-5897) was injected subcutaneously 10 mg/kg for 30 days into adult male langur monkeys (Presbytis). The animals did not lose weight; there was a reduction of weight of the testis and accessories. Histologically, spermatogenesis was suppressed but the interstitial cells appeared normal. In the caput epididymides, the diameter of the tubule was reduced by half as compared to the control and the height of cells was also reduced; in the cauda epididymidis, the cilia were either absent or reduced in size. Sialic acid concentration was reduced in the testis and epididymis of the treated animals; fructose in seminal vesicle and citric acid in prostate were also reduced though morphologically the interstitial cells looked normal, they were functionally anti-anabolic. This appears to be a new profile of alpha-chlorohydrin action in a primate.

Effect of modulating endogenous prolactin secretion on testosterone production in the adult male bonnet monkey (Macaca radiata):

Adult male bonnet monkeys maintained under regulated light: dark conditions exhibit a nycthemeral surge of testosterone. The present study attempts to determine the effect of administration of drugs that modulate prolactin levels like ergobromocriptine (EBC) and chlorpromazine (CPZ) on testosterone production. The injection of EBC, a known inhibitor of prolactin secretion, could abolish nocturnal testosterone surge irrespective of the drug being given at 8.00 or 17.00 h. Testosterone surge could likewise be inhibited by treating animals with CPZ, a potent stimulator of prolactin secretion. This suggests that alteration in endogenous prolactin level from the normal effects nycthemeral surges of testosterone. The in vivo responsiveness of the testes of monkeys injected either CPZ oder EBC to exogenous LH or LHRH stimulation was tested. While LH could completely override the CPZ induced inhibition in testosterone production it could only partially reverse the EBC effect.

Effect of chronic administration of Tamoxifen on fertility in male bonnet monkeys (Macaca radiata).

Administration of Tamoxifen via the Alzet pump at a rate of 50 micrograms hr-1 for 90 days in the adult male bonnet monkeys Macaca radiata had no effect on the serum testosterone concentration determined at 10 AM and 10 PM as well as total sperm count determined at 15-day intervals over a period of 260 days. However, a significant reduction in sperm motility was observed beyond 90 days up until the 225th day. Breeding studies conducted from day 90 to 260 revealed that these males were infertile.

Expression of Nitric Oxide Synthase During Germ Cell Apoptosis in Testis of Cynomolgus Monkey After Testosterone and Heat Treatment

This study investigates the possible involvement of nitric oxide synthase (NOS) in activating germ cell death in monkeys after mild testicular hyperthermia and/or hormonal deprivation. Groups of 8 adult male monkeys received 1 of the following treatments for 12 weeks: 1) 2 empty Silastic implants, 2) 2 testosterone (T) implants, 3) daily exposure of testes to heat (43 degrees C for 30 minutes) for 2 consecutive days, or 4) 2 T implants plus testicular heat exposure. Testicular biopsies were performed before and on days 3, 8, 28, and 84 of the treatment. In control monkey testes, endothelial NOS (eNOS) was observed mainly in Sertoli cells and spermatogonia. No obvious alteration in eNOS levels was detected in any of the treatment group as assessed by Western blotting. Induction of inducible NOS (iNOS) in testes of the 3 treated groups was detected by immunoblotting as early as day 3 after treatment compared with that of controls. Immunocytochemistry further revealed a small increase in iNOS expression in both germ cells and Sertoli cells after T treatment. However, treatment of heat or heat in combination with T markedly induced iNOS expression in germ cells. These data suggest that iNOS, but not eNOS, may be involved in monkey testicular germ cell death after heat and/or T treatment.

Is the positive c-kit immunostaining associated with the presence of cells analogous to the intersticial cells of Cajal in the ciliary muscle?

Interstitial cells of Cajal were identified in the gastrointestinal tract of several species, with close relation to the enteric nervous system. Since it was recognized that interstitial cells of Cajal express the gene product of c-kit, we performed immunohistochemistry for c-kit protein in ciliary muscle specimens of monkeys' eyes. Eight eyes from four adult male new world monkeys (Cebus apella) were studied. After blocking endogenous peroxidase activity and nonspecific protein binding, 1:100 dilution of mouse monoclonal antibody against c-kit human oncoprotein was applied to tissues. Antigen-antibody reaction was visualized using the avidin-biotinylated horseradish peroxidase complex in each slide. We observed some groups of fusiform c-kit expressing cells located amongst muscle bundles of the ciliary muscle. Other pigment cells and mast cells were also observed. C-kit expressing cells observed in the ciliary muscle of Cebus apella, showed no similarity to melanocytes or mast cells and they could be associated with their gastrointestinal interstitial cells of Cajal counterpart.

Immediate loading of orthodontic mini-implants: a histomorphometric evaluation of tissue reaction

Although immediate loading of orthodontic mini-implants can be clinically successful, a lack of histological data exists. The present investigation was performed to evaluate, in an animal model, tissue reaction to immediate loading. Fifty orthodontic titanium mini-implants were inserted in four adult male monkeys at four time intervals. Forty-two devices were loaded with 50 cN super-elastic coil springs immediately after insertion while eight were left unloaded and served as the controls. After euthanasia, the following histomorphometric parameters were evaluated: bone volume (BV/TV), bone-to-implant contact (BIC), mineralizing surface (MS/BS), and erosion surface (ES/BS). Statistical analysis was performed by means of non-parametric tests. Four devices were removed because of loss of stability. A wide variation between animals was found for all parameters. BV/TV: slightly higher values were found in the unloaded sample. Although no particular trend was observed, at 3 months higher values were found in the lower jaw. BIC: a trend to a decrease between 1 week and 1 month followed by a significantly progressive increase was observed. Implants that showed some sections with as little as 3 per cent BIC successfully resisted loading. MS/BS: higher values were found in the lower jaw. MS/BS increased significantly between 1 week and 1 month, followed by a progressive decrease. ES/BS: there was a decrease between 1 week and 1 month, followed by a progressive re-increase. BV/TV did not show any particular trend while BIC was a time-dependent factor. MS/BS and ES/BS demonstrated opposite trends during the healing period. Immediate loading with light forces did not negatively affect the bone healing pattern.

Effects of platelet-rich plasma on healing of alveolar socket: Split-mouth histological and histometric evaluation in Cebus apella monkeys

The prediction of implant treatment is directly influenced by the quality of the remaining bone after tooth extraction. The purpose of this experimental study was to, histologically and histometrically, evaluate the bone repair process in the central areas of extraction sockets filled with platelet-rich plasma. Four young adult male Cebus apella monkeys were used. The extraction of both right and left inferior second premolars was accomplished. After extraction, in one of the extraction sockets, coagulum was maintained while in the other it was removed; the alveolus was dried with gauze compress and filled up with platelet concentrate. For PRP production, Sonnleitner's protocol was followed. The specimens for histological and histometric assessment were obtained in 30, 90, 120 and 180 days intervals. In 30 days new bone formation was intense in both experimental and control sockets and no significant differences were observed between the two groups. After 90 days of the extraction, while the control group showed signs of decrease in osteogenesis, in the experimental unit, the process of bone formation and fibroblast-like cell proliferation remained intense. After 120 days, the PRP treated socket was occupied by large trabeculae of bone. After 180 days, the control unit was occupied mostly with bone marrow. The experimental unit remained occupied with large amounts of bone tissue. It was possible to conclude that bone repair was enhanced by the use of platelet- rich plasma in alveolar sockets.

Predation increases acoustic complexity in primate alarm calls

According to most accounts, alarm calling in non-human primates is a biologically hardwired behaviour with signallers having little control over the acoustic structure of their calls. In this study, we compared the alarm calling behaviour of two adjacent populations of Diana monkeys at Taï forest (Ivory Coast) and Tiwai Island (Sierra Leone), which differ significantly in predation pressure. At Taï, monkeys regularly interact with two major predators, crowned eagles and leopards, while at Tiwai, monkeys are only hunted by crowned eagles. We monitored the alarm call responses of adult male Diana monkeys to acoustic predator models. We found no site-specific differences in the types of calls given to eagles, leopards and general disturbances, but there were consistent differences in how callers assembled calls into sequences. At Tiwai, males responded to leopards and general disturbances in the same way, while at Taï, males discriminated by giving call sequences that differed in the number of component calls. Responses to eagles were identical at both sites. We concluded that Diana monkeys are predisposed to use their repertoire in context-specific ways, but that ontogenetic experience determines how individual calls are assembled into meaningful sequences.

Drinking typography established by scheduled induction predicts chronic heavy drinking in a monkey model of ethanol self-administration.

We have developed an animal model of alcohol self-administration that initially employs schedule-induced polydipsia (SIP) to establish reliable ethanol consumption under open access (22 h/d) conditions with food and water concurrently available. SIP is an adjunctive behavior that is generated by constraining access to an important commodity (e.g., flavored food). The induction schedule and ethanol polydipsia generated under these conditions affords the opportunity to investigate the development of drinking typologies that lead to chronic, excessive alcohol consumption. Adult male cynomolgus monkeys (Macaca fascicularis) were induced to drink water and 4% (w/v in water) ethanol by a Fixed-Time 300 seconds (FT-300 seconds) schedule of banana-flavored pellet delivery. The FT-300 seconds schedule was in effect for 120 consecutive sessions, with daily induction doses increasing from 0.0 to 0.5 g/kg to 1.0 g/kg to 1.5 g/kg every 30 days. Following induction, the monkeys were allowed concurrent access to 4% (w/v) ethanol and water for 22 h/day for 12 months. Drinking typographies during the induction of drinking 1.5 g/kg ethanol emerged that were highly predictive of the daily ethanol intake over the next 12 months. Specifically, the frequency in which monkeys ingested 1.5 g/kg ethanol without a 5-minute lapse in drinking (defined as a bout of drinking) during induction strongly predicted (correlation 0.91) subsequent ethanol intake over the next 12 months of open access to ethanol. Blood ethanol during induction were highly correlated with intake and with drinking typography and ranged from 100 to 160 mg% when the monkeys drank their 1.5 g/kg dose in a single bout. Forty percent of the population became heavy drinkers (mean daily intakes >3.0 g/kg for 12 months) characterized by frequent "spree" drinking (intakes >4.0 g/kg/d). This model of ethanol self-administration identifies early alcohol drinking typographies (gulping the equivalent of 6 drinks) that evolve into chronic heavy alcohol consumption in primates (drinking the equivalent of 16 to 20 drinks per day). The model may aid in identifying biological risks for establishing harmful alcohol drinking.

Social rank and social separation as determinants of alcohol drinking in squirrel monkeys.

Alcohol may be self-administered for its anxiolytic effects to alleviate symptoms of stress, but different types of stressors have varying effects on alcohol intake. Social stress is particularly relevant to alcohol drinking, and a primate model of stress-induced alcohol self-administration would be useful. The objective of the study is to determine if social stresses of different lengths and intensities affect voluntary alcohol intake in monkeys. Subjects were adult male and female squirrel monkeys (Saimiri sciureus) housed in social colonies. Subjects were trained to drink a solution of ethanol and sucrose, alternated daily with a control solution of quinine and an equal concentration of sucrose in 15-min sessions. Drinking was tested during 20-min acute, social separations and 1-week, extended, social separations. Dominance status was quantified using observational records of social interactions within the colonies. Salivary cortisol was sampled in the home colony and during extended social separation. Dominance rank was inversely correlated with alcohol intake during social housing but was not correlated with control fluid intake. Acute social separation abolished drinking of both fluids, accompanied by increased anxiety-like behavior. Extended social separation increased salivary cortisol and alcohol drinking but not control fluid intake in males. In females, drinking was unchanged by extended separation. The chronic stress of social subordination is correlated with increased alcohol drinking. Acute social separation stress suppresses drinking behavior, while extended separation preferentially increases alcohol intake in a subset of individuals. These findings suggest that social stressors of different time-courses and intensities have opposing effects on alcohol self-administration.

Predation on the black capuchin monkey Cebus nigritus (Primates: Cebidae) by domestic dogs Canis lupus familiaris (Carnivora: Canidae), in the Parque Estadual Serra do Brigadeiro, Minas Gerais, Brazil

Predation on an adult male black capuchin monkey, Cebus nigritus (Goldfuss, 1809) by two domestic dogs was observed in the Parque Estadual Serra do Brigadeiro, in the Atlantic Forest of southeastern Minas Gerais. Predation occurred in an area of well preserved native forest 800 m from the nearest forest edge. This is the first confirmed record of predation by domestic dogs in this reserve, yet data from a study in the same area indicates that the domestic dog is the most frequently recorded mammal species, which suggests that it is common in the area. The few published reports indicate that this problem occurs in other conservation units in Brazil and should, therefore, be treated with more rigor by the environmental agencies.

Fatal Disseminated Encephalitozoonosis in a Captive, Adult Goeldi's Monkey (Callimico goeldii) and Subsequent Serosurvey of the Exposed Conspecifics

A captive, adult male Goeldi's monkey (Callimico goeldii) (GM) presented in acute respiratory distress 4 yr after importation into the United States from Europe. Radiographs and echocardiogram were consistent with heart failure. The monkey died within 24 hr of presentation. Necropsy findings included multicentric arteritis and aortitis with aneurysm associated with microsporidian organisms morphologically consistent with Encephalitozoon species. Polymerase chain reaction confirmed organisms were Encephalitozoon cuniculi. Sequence analysis of amplicons generated by using primers specific for the polar tube protein of E. cuniculi determined the organism to be genotype II. An E. cuniculi serosurvey of potentially exposed conspecifics that represented approximately 40% of the captive GM population in the United States was conducted. Multiple individuals that had been imported from Europe with the individual of this report were seropositive via an immunofluorescent antibody assay for E. cuniculi. Multiple samples were available from 3 individuals that demonstrated a decrease in titer or reversion to seronegative status within 3 yr of initial positive status. All other GM were negative on serology. This case is unique in that the genotype identified (genotype II) was different than the genotype (genotype III) reported in other New World primate (NWP) species, the patterns of arteritis were different from the typical pattern of microsporidial vasculitis described in other species, and clinical disease was observed in an adult. Most reported cases of clinical disease secondary to E. cuniculi in NWP have been in neonates and juveniles.

Characterization of dopamine D1, D2, and D3 receptor function in adult rhesus monkeys exposed to cocaine in utero

In fetal rhesus monkeys, prenatal cocaine exposure has been shown to increase dopamine (DA) D1 and D2 receptor densities within the striatum (Fang et al., 1997), but little is known about the long‐term effects of prenatal cocaine exposure on the DA system. In the present study, D2 receptor availability in the striatum, as measured by positron emission tomography (PET), was not different in adult rhesus monkeys that had been exposed to cocaine (n=10) in utero (Morris et al., 1996) compared to controls (n=8). The ability of a high‐efficacy D1 agonist, SKF 81297 (0.03–0.3 mg/kg), to elicit blinking was then characterized. Preliminary data (n=3) indicates that there is a dose‐dependent increase in blinking, with cocaine‐exposed monkeys appearing slightly more sensitive than control monkeys. To study the D3 receptor system, quinpirole‐induced yawning was examined. The prenatally cocaine exposed male monkeys exhibited increased sensitivity to the stimulating effects of quinpirole compared to controls. The prenatally cocaine exposed female monkeys did not differ from controls. These preliminary data suggests that gestational cocaine alters DA receptor function in adult male monkeys. This is particularly striking when one considers the prenatal exposure was over 13 years ago. Whether these neurochemical changes influence the behavioral vulnerability to self‐administer cocaine remains to be studied. DA 10584

Modulation of food intake following deep brain stimulation of the ventromedial hypothalamus in the vervet monkey

Deep brain stimulation (DBS) has become an effective therapy for an increasing number of brain disorders. Recently demonstrated DBS of the posterior hypothalamus as a safe treatment for chronic intractable cluster headaches has drawn attention to this target, which is involved in the regulation of diverse autonomic functions and feeding behavior through complex integrative mechanisms. In this study, the authors assessed the feasibility of ventromedial hypothalamus (VMH) DBS in freely moving vervet monkeys to modulate food intake as a model for the potential treatment of eating disorders. Deep brain stimulation electrodes were bilaterally implanted into the VMH of 2 adult male vervet monkeys by using the stereotactic techniques utilized in DBS in humans. Stimulators were implanted subcutaneously on the upper back, allowing ready access to program stimulation parameters while the animal remained conscious and freely moving. In anesthetized animals, intraoperatively and 6-10 weeks postsurgery, VMH DBS parameters were selected according to minimal cardiovascular and autonomic nervous system responses. Thereafter, conscious animals were subjected to 2 cycles of VMH DBS for periods of 8 and 3 days, and food intake and behavior were monitored. Animals were then killed for histological verification of probe placement. During VMH DBS, total food consumption increased. The 3-month bilateral implant of electrodes and subsequent periods of high-frequency VMH stimulation did not result in significant adverse behavioral effects. This is the first study in which techniques of hypothalamic DBS in humans have been applied in freely moving nonhuman primates. Future studies can now be conducted to determine whether VMH DBS can change hypothalamic responsivity to endocrine signals associated with adiposity for long-term modulation of food intake.

Reversible medetomidine/ketamine anesthesia in captive capuchin monkeys (Cebus apella)

Medetomidine, an alpha(2)-adrenergic receptor agonist administered with ketamine, induces sedation/anesthesia in non-human primates in a dose-dependent, species-specific manner. Two adult male capuchin monkeys enrolled in a study of kinematics of mastication were administered intramuscular (IM) injections of 150, 200 and 400 microg/kg medetomidine or medetomidine (microg/kg)/ketamine (mg/kg) at 200/2, 150/4. Medetomidine was reversed with atipamezole. Animals were then fed for 9-75 minutes. A second anesthesia reversal episode allowed return to home cage. Medetomidine (150 microg/kg)/ketamine (4 mg/kg) provided anesthesia for handling and restraint within 10 minutes of administration. Atipamezole (750 microg/kg) IM provided reversal sufficient for the animal to eat by 22 minutes or perch by 31 minutes. Medetomidine/ketamine administered IM in Cebus apella results in rapid, reproducible and safe anesthesia which is reversible with atipamezole. Nausea, vomiting or regurgitation were not observed during anesthesia reversal episodes in either the fasted or fed state.

Effect of deprivation of endogenous follicle stimulating hormone on rat epididymis: a histological evaluation

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50–60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or flutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

Selective PDE inhibitors rolipram and sildenafil improve object retrieval performance in adult cynomolgus macaques

RationaleSelective phosphodiesterase (PDE) inhibitors improve the formation of hippocampus-dependent memories in several rodent models of cognition. However, studies evaluating the effects of PDE inhibition on prefrontal cortex-dependent cognition and in monkeys are rare.ObjectivesThe present study investigates the effect of the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil on object retrieval performance. Object retrieval is a prefrontal cortical-mediated task, which is likely to capture attention and response inhibition.Materials and methodsThe ability to retrieve a food reward from a clear box with an open side positioned in various orientations was assessed in adult male cynomolgus monkeys (Macaca fascicularis).ResultsRolipram (0.003–0.03 mg/kg, intramuscular [i.m.]) and sildenafil (0.3–3 mg/kg, i.m.) dose-dependently increased correct first reaches during difficult trials, reaching significance at 0.01 and 1 mg/kg, respectively. For both drugs, correct reaches were increased approximately 20%; that is, performance was improved from ~50 to ~70% correct.ConclusionsBoth rolipram and sildenafil improved object retrieval performance, thus demonstrating the cognition-enhancing effects of PDE inhibition on a prefrontal task of executive function in monkeys.

Multiple Granulomas in Three Squirrel Monkeys ( Saimiri sciureus ) Caused by Mycobacterium microti

Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, causes tuberculosis in small rodents and occasionally in other mammals including man. Three adult male squirrel monkeys, two with a history of lethargy, weakness and stridor and one with paralysis of the hind legs, were presented for necropsy. One of the two lethargic animals showed multiple granulomas in the mesentery, mesenteric lymph nodes, lung, liver, kidneys and spleen, while the other showed granulomas only in the lung. The animal with paralysis of the legs had an abscess-like lesion in the skeletal muscle of the neck, granulomas in the mesenteric and mediastinal lymph nodes, and a fracture of the thirteenth thoracic vertebra with severe lesions of the spinal cord. Histologically the granulomas showed typical features of tuberculous granulomas, i.e., central necrosis surrounded by epithelioid cells, multinucleated giant cells, inflammatory cells and a border of connective tissue. Ziehl-Neelsen stain demonstrated sporadic acid-fast bacilli within the granulomas, these organisms being identified as M. microti by microbiological and molecular methods.

Angiotensin II AT 1 receptor blockade normalizes CD11b + monocyte production in bone marrow of hypercholesterolemic monkeys

The enhanced production of monocytes expressing pro-inflammatory markers such as the integrin CD11b in patients with hypercholesterolemia may promote vascular inflammation and exacerbate atherogenesis. The objective of the present study was to determine whether hypercholesterolemia stimulates the production of CD11b + monocytes in bone marrow, and whether the renin–angiotensin system participates in this process and thus provides a target for therapeutic intervention. The dietary induction of hypercholesterolemia in adult male cynomolgus monkeys was accompanied by increased bone marrow cellularity and elevated peripheral blood and bone marrow monocyte CD11b expression. Isolated bone marrow CD34 + hematopoietic stem cells (HSCs) evaluated by in vitro functional assays exhibited enhanced myeloproliferative capacity and differentiation into CD11b + monocytes. Treatment of hypercholesterolemic monkeys with the angiotensin II AT 1 receptor blocker losartan for 15 weeks reduced bone marrow cellularity, suppressed peripheral blood and bone marrow monocyte CD11b expression, and normalized CD34 + cell function assays. All variables returned to pretreatment levels 6 weeks after discontinuation of losartan treatment. Hypercholesterolemia was associated with increased CD34 + cell AT 1 receptor expression and an exaggerated in vitro myeloproliferative response to angiotensin II stimulation that positively correlated to plasma LDL concentrations. In vitro exposure to native low-density lipoproteins (LDL) also increased CD34 + cell AT 1 receptor expression and the myeloproliferative response to angiotensin II stimulation in a dose-dependent and receptor-mediated manner. Our data provide support for a positive regulatory role of plasma LDL on AT 1 receptor-mediated HSC differentiation and the production of pro-atherogenic monocytes. LDL-regulated HSC function may explain in part hypercholesterolemia-induced inflammation as well as the anti-inflammatory and anti-atherosclerotic effects of AT 1 receptor blockers.

Effects of levonorgestrel butanoate alone and in combination with testosterone buciclate on spermatogenesis in the bonnet monkey

The spermatogenic effects of levonorgestrel butanoate were studied in adult male bonnet monkeys when administered alone and in combination with testosterone buciclate. Levonorgestrel butanoate (0.25, 1.0 and 2.5 mg kg(-1)) given as two injections on days 0 and 60 (groups II, III, IV) resulted in thickening and folding of the basement membrane and disruption of cell associations in groups III and IV (on day 120). In group II, no apparent changes in testicular histology were observed. When these doses of levonorgestrel butanoate were combined with 40 mg of testosterone buciclate (groups V, VI, VII), maximum changes were seen in group VI in which all stages of spermatogenesis were absent on day 120 except for a small number of spermatogonia. The changes caused by lower dose (group V) and higher dose (group VII) of levonorgestrel butanoate were less prominent than in group VI. A significant decrease in the number of dark A (Ad) and B spermatogonia was observed in all groups except for Ad spermatogonia on day 120 in group V, B spermatogonia on day 60 in group IV and B spermatogonia on day 120 in group III. A significant decrease in pachytene spermatocytes was seen on day 120 in groups V only. Early spermatids showed a significant decrease only in groups V and VII on day 120 of treatment. Advanced spermatids were suppressed significantly in group IV on day 60 and in groups IV and V on day 120. These data indicate that levonorgestrel butanoate (1.0 mg kg(-1)) in combination with 40 mg of testosterone buciclate was the most effective treatment in suppressing spermatogenesis. The site of action of this combination regimen is at the level of renewing Ad spermatogonia.