Characterization and Localization of Membrane Vesicles in Ejaculate Fractions from the Ram, Boar and Stallion

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (+/- s.e.m.) vesicle diameters were 130.9 +/- 3.22 (range 18-577), 164.1 +/- 4.42 (range 15-671) and 159.7 +/- 2.92 nm (range 22-986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi-laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule-dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.     

Bilateral seminal vesiculitis and ampullitis in a stallion

A Thoroughbred stallion suspected of having venereal disease was found to have an infection of the accessory sex glands. Purulent debris, blood, and Pseudomonas aeruginosa were recovered from all ejaculates. Treatment with gentamicin sulfate, tobramycin, and amikacin sulfate was unsuccessful in eliminating the infection. The stallion's seminal plasma, collected during treatment with gentamicin sulfate, did not contain any appreciable antibacterial activity. Apparently, negligible amounts of gentamicin diffused across the mucosal cell borders of the accessory sex glands into the seminal plasma. Bilateral seminal vesiculitis and ampullitis were evident on examination of the reproductive tract when the stallion was euthanatized.     

Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen–Thawed Ram Spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination.     

Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa

Ram seminal plasma increases the fertility of frozen-thawed ram spermatozoa deposited into the cervix. The aim of the current study was to compare the effect of ram seminal plasma to that of bull seminal plasma, dog prostatic fluid, protein-free TALP TrilEq (Triladyl with 0.5 mt of Equex STM paste added to each 100 mt) and heat-treated skim milk on longevity and percentages of progressively motile and aberrantly motile frozen-thawed ram spermatozoa. Three ejaculates from each of 6 rams were extended in TrilEq, pooled and frozen in straws as a single batch per ram. One hundred and eight straws (3 straws from each ram for each fluid) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 ml of the appropriate fluid at 37 degrees C and kept at that temperature for 6 h. Motility was assessed at x200 magnification immediately (time zero) and 2, 4 and 6 h after thawing. Progressive motility decreased from each time to the next (P < 0.05) and was 39.0 % (0 h), 26.0 % (2 h), 19.6 % (4 h) and 12.6 % (6 h); SEM 1.24, n = 108 for each group. Ram seminal plasma resulted in higher progressive motility than bull seminal plasma, lower than milk, and similar to the other fluids. Ram seminal plasma resulted in lower aberrant motility than protein-free TALP and similar aberrant motility to other fluids. The effect of ram seminal plasma and dog prostatic fluid was very similar. The effect of ram seminal plasma on the fertility of frozen-thawed ram spermatozoa deposited into the cervix is not due an exceptionally beneficial effect on the motility of spermatozoa.     

Gene and protein expression in the reproductive tract of Brazilian Somalis rams

Brazilian Somalis is a locally‐adapted breed of rams raised in tropical climate and native pastures. The present study was conducted to evaluate gene expression and proteome of the reproductive tract of such rams. Samples were collected from testes, epididymides, seminal vesicles and bulbourethral glands of four rams. Expression of clusterin (CLU), osteopontin (OPN) and prostaglandin D2 synthase (PGDS) genes were evaluated in all samples by real‐time PCR. Shotgun proteomic analysis was performed using samples from the head, corpus and cauda epididymides and from all other structures as well. Gene ontology terms and protein interactions were obtained from UniProtKB databases and MetaCore v.6.8 platform. CLU trasncripts were detected in the testes, epididymides, seminal vesicles and bulbourethral glands of the Somalis rams. The initial region and body of the epididymis had the greatest CLU expression. OPN mRNA was localized in all tissues of the ram reproductive tract. PGDS mRNA was detected in the testes and epididymides. Lable‐free mass spectrometry allowed the identification of 137 proteins in all samples. Proteins of the epididymis head mainly participate in cellular processes and response to stimulus, participating in catalityc activity and binding. Proteins of epididymis body acted as regulatory proteins and in cellular processes, with binding and catalytic activity. Cauda epididymis molecules were associated with cellular processes and regulation, with binding function and catalytic activity as well. Testis proteins were mainly linked to cell processes and response to stimuli, and had catalytic function. Seminal vesicle proteins were involved in regulation and mainly with binding functions. Most bulbourethral gland proteins participated in cellular processes. The present study is the first to evaluate the proteome and gene expressions in the reproductive tract of Brazilian Somalis rams. Such pieces of information bring significant cointribution for the understanding of the reproductive physiology of locally‐adapted livestock.     

Immunoglobulins and immunoglobulin-containing cells in the reproductive tract of normal rams

The levels of the immunoglobulins IgA, IgG1, IgG2, and IgM were measured in serum and fluid from various locations in the reproductive tract of normal rams. These fluids included semen, preputial washings, and fluid from the accessory sex glands (ASG), vasa deferens, rete testes, and tissue fluid from the seminal vesicles, bulbourethral glands, epididymal tails and efferent ducts. In addition, the prevalence of specific Ig-containing cells (ICC) was measured in sections of formalin fixed tissues stained by an indirect peroxidase-antiperoxidase labelling technique. Mean IgA levels in semen (1.23 mg/ml) and ASG fluid (0.46 mg/ml), were higher than in serum (0.19 mg/ml) and were at levels higher than IgG1 or IgG2 levels in semen, ASG fluid, and preputial washings, thus confirming the existence of a local immune system primarily in the ASG of ram genitalia. Relatively low concentrations of IgA and IgG in other genital fluids and IgG levels in these fluids were consistent with diffusion from serum. The relatively high prevalence of IgA-containing cells in bulbourethral (56% of all ICC) and prostate (49%) glands confirmed these tissues as major sites of local Ig production. ICC were also found in large numbers beneath pelvic urethral and preputial epithelia, but these were predominantly IgG-containing (88 and 72% respectively).     

Immunoglobulins and immunoglobulin-containing cells in the reproductive tracts of rams naturally infected with Brucella ovis

SUMMARY Thirteen rams with serological evidence of Brucella ovls exposure (CFT of 1:8 or greater), but with no or only mild epididymitis, were selected from a ram flock. Serum, semen, preputial washings and fluids from the accessory sex glands (ASGF) and testis and epididymis (TEF) were examined and immunoglobulin (lg) concentrations estimated. Genital tissues were examined histologically and the percentages of class specific immunoglobulin containing cells (ICC) determined. Eleven of these rams had histological evidence of active inflammation consistent with B. ovis infection; the organisam was cultured from the semen of 7. IgA concentration was high in semen (mean ± standard deviation of 5.03 ± 1.78 mg/ml) and ASGF (9.18 ± 7.28 mg/ml). These levels were much higher than those recorded in noninfected rams. IgA concentration was low in serum (0.78 ± 0.55 mg/ml) and TEF (0.59 ± 0.78 mg/ml). The concentrations of IgG¹, IgG², and IgM were low in all genital fluids sampled and not significantly different from those recorded in noninfected rams. This indicated that infection with B. ovis results in a pronounced IgA response in secretions, mostly from the accessory sex glands. Examinations of ICC, however, revealed that the plasma cell infiltrates of the epididymis, vas deferens, ampulla and seminal vesicle were predominantly IgG-containing (92.4, 97.2, 79.4 and 91.9% respectively). Fewer IgM-containing cells were scattered throughout these tissues, constituting 3.9, 6.3, 0.3 and 6.5% of all ICC, respectively. IgA-containing cells were most frequently seen in the ampulla (9.6% of ICC) where they were located directly beneath the epithelium, suggesting the ampulla as the most prominant location for the local production of IgA. These results indicate that although the tissue ICC response was predominantly IgG, IgA was selectively transferred into secretions with the exclusion of most IgG and IgM. ICC percentages in bulbourethral and prostate glands, and beneath pelvic urethral and preputial epithelia, were similar to those found in noninfected rams.     

Isolation of Actinobacillus seminis from rams in the United Kingdom.

Actinobacillus seminis was isolated from the semen of five rams on four farms. Four of the rams had abnormal semen and three were also infertile. The isolates of A seminis showed similar phenotypic profiles and electrophoretic protein patterns to the type strain of A seminis but were distinct from Histophilus ovis previously isolated from rams with epididymitis in Scotland. The infection appeared to be subclinical but two of the five rams had palpable abnormalities of their testes. Three rams were treated with antibiotics but the infection persisted. No gross lesions were found in the genitalia of two of three rams examined post mortem but one had necrotic abscesses in the testes and epididymis. A seminis was isolated from the seminal vesicles and epididymis of one ram without gross lesions but not from the genitalia of the other two. On one farm the infection in a recently purchased ram led to the detection of another case as a result of the bacteriological screening of 11 stock rams not in contact with the initial case. These five subclinical cases, which included a supposedly healthy stock ram, suggest that A seminis infection may be widespread and should be considered in cases of infertility.     

Effects of vasectomy on fructose levels in the ejaculate of rams.

Fructose concentration was studied in the ejaculates of 20 intact and 19 vasectomised rams over a two year period. In intact rams the concentration ranged from 222-740 mg/100 ml of seminal plasma during the breeding season (October to December), with lower values (0-300 mg/100) during the rest of the year. In vasectomised rams the seasonal pattern was less marked with very high values (800-1260 mg/100 ml) as well as low values (0-20 mg/100 ml) being encountered during both the breeding and non-breeding seasons, indicating changes in accessory gland function.     

Electroejaculation Increases Low Molecular Weight Proteins in Seminal Plasma Modifying Sperm Quality in Corriedale Rams

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.     

A STUDY OF THE ACCESSORY SEX GLANDS OF BULLS EN ABATTOIRS IN NORTHERN AUSTRALIA

The accessory sex glands of 521 slaughtered bulls were examined for lesions; normal anatomical observations were also made.
The average weights and measurements (length and width) of the seminal vesicles and bulbourethral glands of 110 bulls were 34.1 g and 9.1 × 2.5 cm, and 4.6 g and 2.9 × 1.9 cm, respectively. Weights of both glands were correlated to age, but bulbourethral gland weight was more closely correlated to bodyweight than age. The average length and diameter of the ampulla was 11.1 and 1.0 cm, respectively.
Seminal vesiculitis and ampullitis were found in 9.0% and 3.8% of bulls respectively. The incidence of both conditions was highest in old bulls. Mesonephric duct anomalies (observed in 1.7% of bulls) and other congenital and inflammatory lesions of the accessory sex glands were also recorded.
Macroscopically normal accessory sex glands of 86 bulls were studied histologically and a high percentage of all glands showed inflammatory changes.
Attempts to isolate viral agents, Mycoplasma, and Chlamydia from accessory sex glands were unsuccessful. Corynebacterium pyogenes was isolated from two cases of seminal vesiculitis. IBR-IPV virus, Campylobacter fetus subsp. venerealis and Tritrichomonas foetus were frequently isolated from the prepuce of bulls, many of which also had positive serological titres to Leptospira pomona and Leptospira hardjo.
No one factor capable of causing bull infertility was consistently identified, and the incidence figures for observed conditions of the accessory sex glands were generally comparable to those found in bull populations elsewhere.     

Immunoglobulin containing cells in normal and inflamed accessory sex glands of bulls

The peroxidase-antiperoxidase (PAP) technique was used to identify cytoplasmic immunoglobulins in the accessory sex glands of 15 normal bulls and 13 bulls with inflammation of the ASG. Immunoglobulin containing cells (ICC) of the types IgA, IgM, total IgG, IgG1 and IgG2 were measured and their percentages expressed. In accessory sex glands from normal bulls, IgA containing cells were the most frequent in prostate and bulbourethral glands (86.7% and 86.1%, respectively of all ICC present) whereas in the ampulla, IgG containing cells comprised 78.6% of the ICC. IgG1 and IgG2 containing cells were present in all the accessory sex glands in approximately equal numbers. Frequencies of IgM containing cells in the ampulla, prostate and bulbourethral glands were 6.3%, 4.0% and 3.7%, respectively. Although all isotypes of ICC were present in the seminal vesicle, the very low number precluded accurate quantification. In inflamed ampulla, seminal vesicle, bulbourethral gland and colliculus seminalis, IgG containing cells were the most frequent ICC with values of 66.2%, 83.0%, 69.0% and 53.5%, respectively; IgA containing cells were the second in prevalence with values of 21.5%, 10.3% 19.3% and 40.5%, respectively. The contribution of ICC to the locally protective immunoglobulins in accessory sex gland secretions is discussed.     

The effect of exogenous administration of oestrogens on the function of the epididymis and the accessory sex glands in the boar.

Three normal, sexually mature young boars of Swedish Yorkshire breed were treated with daily intramuscular injections of diethylstilboestrol and/or oestradiolbenzoate for a total period of 13–15 weeks. Whole ejaculates were collected on a dummy sow with an artificial vagina twice a week. The ejaculate was examined as regards semen volume, sperm concentration, sperm motility and sperm morphology. The seminal plasma was analysed for Na, K, Cl, Mg, inorganic phosphate, total protein and fructose. Testicles, epididymides and the accessory sex glands were obtained at slaughter. Material from epididymal segments A, B, D_b, F_a and F_b was collected and examined for sperm morphology. Spermatocrit, osmotic pressure and plasma concentrations of Na, K, Gl, total protein and GPC were assessed in material from epididymal segment F_a. The sex glands and the accessory sex glands were examined maeroscopically and microscopically.The semen volume, the sperm concentration and consequently the total sperm count per ejaculate showed a gradual increase during the course of the investigation. This is to be expected as the boars used were comparatively young at the beginning of the experiment. These values were all within normal limits for adolescent boars.Taking all the characteristics examined into consideration no conclusive evidence was found that an exogenic administration of oestrogens to intact boars has any influence on the function of the epididymis and the accessory sex glands.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Effect of method of seminal collection on the retrograde flow of spermatozoa into the urinary bladder of rams

The effects of method of seminal collection and a diuretic on retrograde flow of spermatozoa into the urinary bladder of rams were examined. In experiment 1, semen and urine were collected from 8 rams during the non-breeding season. Prior to seminal collection, all rams were given furosemide and a sample of urine was obtained during micturition. Semen was then collected from each ram with an artificial vagina or by electroejaculation in alternate weeks for 4 weeks, and the urine released during the first postseminal collection micturition was collected in 4 consecutive samples. The volume of electroejaculates was larger (P less than 0.0001) than the volume of ejaculates, but the total number of spermatozoa in the electroejaculate or in the ejaculate were not different (P greater than 0.1). Urine obtained before seminal collection was azoospermic or contained few, nonmotile spermatozoa (mean +/- SD = 0.053 +/- 0.114 x 10(6)/ml). The adjusted spermatozoal concentration (mean +/- SD = 1.630 +/- 2.258 x 10(6)/ml) in the urine collected after seminal collection was 31 times higher (P less than 0.0001) and there were motile spermatozoa in most (97%) of the samples. The spermatozoal concentration in sequential samples of urine was not different (P greater than 0.1) between samples and was not affected (P greater than 0.1) by the method of seminal collection. There was a trend, approaching significance (P = 0.052), for an effect of method of seminal collection on the percentage of retrograde flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of male accessory gland secretions on sensitivity of porcine sperm acrosomes to cold shock, initiation of motility and loss of cytoplasmic droplets

Hyaluronidase release was used as an index of acrosomal membrane damage during cold shock of epididymal boar sperm and ejaculated sperm from intact and vesiculectomized boars. Sperm were also incubated with seminal plasma from intact and vasectomized boars to examine the contributions of male accessory gland secretions. Acrosomal membranes of epididymal sperm were more resistant to cold shock than those of ejaculated sperm. Only 36% of the hyaluronidase released by ejaculated sperm was released by the epididymal sperm in spite of similar hyaluronidase content of the sperm. Preincubation of epididymal sperm in seminal plasma from both intact and vasectomized boars increased resistance to cold shock by 60 to 80%. Initial dilution of epididymal sperm with seminal plasma, rather than Ringer-fructose buffer, was associated with low progressive motility and with retention of cytoplasmic droplets. In contrast, acrosomal membranes of ejaculated sperm from intact and vesiculectomized boars exhibited similar sensitivity to cold shock, releasing hyaluronidase capable of forming .20 and .19 mumol N-acetylglucosamine from hyaluronic acid/10(8) sperm in 8 min. Moreover, seminal plasma from vasectomized boars had no effect on acrosomal sensitivity to cold shock of ejaculated sperm from vesiculectomized boars.     

Seasonal changes in reproductive parameters and seminal plasma constituents of rams in Afyon province of Turkey

The objective of the present study was to describe seasonal changes in semen quality, serum testosterone and seminal plasma constituents for 9 months in 10 rams from a mature local (Daglic) and imported breed (Chios) under Afyon province conditions typical for the internal Anatolian region. Sperm motility and sperm concentration and testosterone level were higher, percentage of abnormal spermatozoa was the lowest during autumn. The biochemical analyses of ram seminal plasma indicated that the highest values of total protein, albumin (A), globulin (G), total lipid and cholesterol were recorded in autumn while A/G ratio exhibited the lowest values. Aspartate amino transferase (AST) activity and AST/ALT ratio recorded the lowest values, while moderate values were recorded for alanine amino transferase (ALT) activity in autumn. The results suggest that the rams under Afyon province conditions show seasonal changes for some reproductive parameters and seminal plasma constituents.     

Serological and necropsy findings for rams infected with Brucella ovis which were not identified by the complement fixation test

The eradication of Brucella ovis from a commercial flock of 36 Romney rams was complicated by four infected rams remaining undetected despite four successive flock examinations using the complement fixation test. These four rams were subsequently tested using an enzyme-linked immunosorbent assay and a gel diffusion test and shown to be infected by semen culture. All four rams could have been identified as infected at the initial test if the enzyme-linked immunosorbent assay had been used in addition to the complement fixation test. Although gross evidence of epididymitis was found in only one ram at necropsy, three had histological lesions of epididymitis and all four had a seminal vesiculitis.     

Reproductive Development of Santa Inês Rams During the First Year of Life: Body and Testis Growth,Testosterone Concentrations,Sperm Parameters,Age at Puberty and Seminal Plasma Proteins

We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two‐dimensional SDS‐PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 ± 0.7 to 54.3 ± 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non‐significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 ± 0.05 to 1.14 ± 0.24 × 10⁹ cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 ± 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 ± 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands.