murrah buffalo bulls were divided into two groups : g1 contained four 18-month - old males , 
 and g2 contained three 24-month - old males . the animals were obtained from the department 
 of animal production , faculty of veterinary medicine , unesp - botucatu / sp , which is located 
 at 2253'09 " s , 4826'42 " w and is 804 m above sea level . the buffalo were fed 
 brachiariasp . and mineral salt ad libitum and 
 supplemented with corn silage during the dry periods . endoparasites control was performed 
 with levamisole phosphate , applied 1 ml per 40 kg of liveweight , and the vermifuge 
 ( ripercol , fort dodge animal health , campinas , so paulo , brazil ) was administered to the 
 animals at the beginning of the wet season ( november ) and early dry season ( june ) . the 
 vaccines were applied to the animals : the first dose of clostridial was given at four 
 months , with a booster after one month followed by subsequent annual applications ; fmd was 
 administered in may and november . the buffalo used in this study were the same age at the beginning of the experiment . the 
 testes were collected at two time points because the specific age of onset of puberty and 
 spermatogenesis in murrah buffalo bulls raised under the conditions described was 
 unknown . the project was conducted in accordance with the ethical principles for animal 
 experimentation of the animal experimentation ethics committee of the school of animal 
 science of unesp dracena ( protocol no . 017/2011 ) . prior to collecting the testes , the animals were physically restrained and anesthetized 
  according to the ethical principles for 
 animal experimentation as recommended by the brazilian college of animal experimentation 
 , and they were weighed using a mechanical 
 balance ( acr 1500 , 4,000 kg , balanas ares , camb , pr , brazil ) . after collection , the epididymis was separated from the testis , and the testicular weight 
 ( tw ) , testicular length ( tl ) , testicular width ( twd ) , testicular thickness ( tt ) and 
 testicular circumference ( tc ) were determined for each testis . to obtain the tw ( g ) , an 
 analytical balance was used ( bk 3000 ; range 0.23100 g ) . each testis was sectioned into three regions ( the capitata , middle and end caudata 
 portions ) for tissue fixation . the middle portions of the testicular parenchyma were 
 placed in bouin 's solution for at least 24 h. after dehydration in increasing 
 concentrations of alcohol , followed by infiltration with xylenes , the tissue samples were 
 embedded in paraffin . a microtome ( leica 
 rm2145 , leica , berlin , germany ) was used to section the tissue into 5-mm - thick slices , and 
 these sections were stained with hematoxylin- and eosin ( he ) . the samples were analyzed 
 under a light microscope ( leica dm 2500 , leica ) . descriptive analyses of the mean and standard deviation for each testicular biometric 
 parameter were performed with the graphpad prism4 software ( graphpad software , la jolla , 
 ca , usa ) . age , body weight and testicular biometric parameters were tested for 
 correlations using r statistical software ( r development core team 2009 ) . murrah buffalo bulls were divided into two groups : g1 contained four 18-month - old males , 
 and g2 contained three 24-month - old males . the animals were obtained from the department 
 of animal production , faculty of veterinary medicine , unesp - botucatu / sp , which is located 
 at 2253'09 " s , 4826'42 " w and is 804 m above sea level . the buffalo were fed 
 brachiariasp . and mineral salt ad libitum and 
 supplemented with corn silage during the dry periods . endoparasites control was performed 
 with levamisole phosphate , applied 1 ml per 40 kg of liveweight , and the vermifuge 
 ( ripercol , fort dodge animal health , campinas , so paulo , brazil ) was administered to the 
 animals at the beginning of the wet season ( november ) and early dry season ( june ) . the 
 vaccines were applied to the animals : the first dose of clostridial was given at four 
 months , with a booster after one month followed by subsequent annual applications ; fmd was 
 administered in may and november . the buffalo used in this study were the same age at the beginning of the experiment . the 
 testes were collected at two time points because the specific age of onset of puberty and 
 spermatogenesis in murrah buffalo bulls raised under the conditions described was 
 unknown . the project was conducted in accordance with the ethical principles for animal 
 experimentation of the animal experimentation ethics committee of the school of animal 
 science of unesp dracena ( protocol no . 017/2011 ) . prior to collecting the testes , the animals were physically restrained and anesthetized 
  according to the ethical principles for 
 animal experimentation as recommended by the brazilian college of animal experimentation 
 , and they were weighed using a mechanical 
 balance ( acr 1500 , 4,000 kg , balanas ares , camb , pr , brazil ) . after collection , the epididymis was separated from the testis , and the testicular weight 
 ( tw ) , testicular length ( tl ) , testicular width ( twd ) , testicular thickness ( tt ) and 
 testicular circumference ( tc ) were determined for each testis . to obtain the tw ( g ) , each testis was sectioned into three regions ( the capitata , middle and end caudata 
 portions ) for tissue fixation . the middle portions of the testicular parenchyma were 
 placed in bouin 's solution for at least 24 h. after dehydration in increasing 
 concentrations of alcohol , followed by infiltration with xylenes , the tissue samples were 
 embedded in paraffin . a microtome ( leica 
 rm2145 , leica , berlin , germany ) was used to section the tissue into 5-mm - thick slices , and 
 these sections were stained with hematoxylin- and eosin ( he ) . the samples were analyzed 
 under a light microscope ( leica dm 2500 , leica ) . descriptive analyses of the mean and standard deviation for each testicular biometric 
 parameter were performed with the graphpad prism4 software ( graphpad software , la jolla , 
 ca , usa ) . age , body weight and testicular biometric parameters were tested for 
 correlations using r statistical software ( r development core team 2009 ) . at 18 months , the testicular parenchyma contained testicular cords and internal cord 
 compartments associated with the presence of leydig cells , vessels and nerves . within the 
 testicular cords , gonocytes , undifferentiated supporting cells and luminal processes were 
 observed . gonocytes and spermatogonia in the differentiation process were identified near 
 the basal membrane ( fig . 1 . testis of murrah buffalo bulls at 18 months of age characterized by an immature 
 sexual phase ( a and b ) . we observed solid testicular cords with gonocytes ( a ) and 
 undifferentiated supporting cells ( s ) . although spermatids were present in high numbers at 18 months in nili - ravi 
 buffalo testes , spermatozoa were not present 
 in the tubule lumens analyzed here . testis of murrah buffalo bulls at 18 months of age characterized by an immature 
 sexual phase ( a and b ) . we observed solid testicular cords with gonocytes ( a ) and 
 undifferentiated supporting cells ( s ) . the testicular parenchyma at 24 months contained seminiferous tubules with lumens and 
 germinative epithelia present at different stages of the seminiferous epithelial cycle 
 ( fig . 2 . different cell types in the seminiferous epithelium of the murrah buffalo at 24 
 months of age . photomicrographs of cross sections of the seminiferous tubules 
 represent the stages of the cycle of the seminiferous epithelium . stage one ( a ) , two 
 ( b ) , three ( c ) , four ( d ) , five ( e ) , six ( f ) , seven ( g ) and eight ( h ) represent the 
 following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno ( pl ) 
 stage , primary spermatocytes at the pachytene ( p ) stage , round spermatids ( ar ) , 
 elongated spermatids ( al ) , primary spermatocytes at the zygotene ( z ) stage , primary 
 spermatocytes at the diplotene ( d ) stage , residual bodies ( cr ) and spermatozoa ( e ) . all the stages presented spermatogonia type a ( a ) and sertoli cells ( s ) . 
 hematoxylin - eosin staining . ) . the following cell types were identified : sertoli cells , spermatogonia , 
 pre - leptotene / leptotene spermatocytes , pachytene spermatocytes , zygotene spermatocytes , 
 diplotene spermatocytes , round spermatids and luminal spermatozoa ( fig . these cells could be classified into eight stages of 
 cellular associations according to the tubular morphology method . bongso   noted that the 
 spermatozoa in the tubular lumen of swamp buffalo had the same morphology at 16 months . in 
 crossbred buffalo ( mediterranean  jaffarabadi ) , this morphology was observed at 13 months 
 . ohashi   showed the onset of the spermatogenic process in buffalo at 9 months . at 24 
 months , our histological analysis of the spermatogenic process indicated 
 that these animals were not sexually mature .   different cell types in the seminiferous epithelium of the murrah buffalo at 24 
 months of age . photomicrographs of cross sections of the seminiferous tubules 
 represent the stages of the cycle of the seminiferous epithelium . stage one ( a ) , two 
 ( b ) , three ( c ) , four ( d ) , five ( e ) , six ( f ) , seven ( g ) and eight ( h ) represent the 
 following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno ( pl ) 
 stage , primary spermatocytes at the pachytene ( p ) stage , round spermatids ( ar ) , 
 elongated spermatids ( al ) , primary spermatocytes at the zygotene ( z ) stage , primary 
 spermatocytes at the diplotene ( d ) stage , residual bodies ( cr ) and spermatozoa ( e ) . all the stages presented spermatogonia type a ( a ) and sertoli cells ( s ) . 
 hematoxylin - eosin staining . mean * body and testicular biometric parameters in murrah buffalo bulls at 18 
 and 24 months of ageage groupbw ( kg)sc ( cm)tw ( cm)tl ( cm)twd ( cm)tt ( cm)tc ( cm)g1 ( 18 mo)270.25  14.5918.75  1.521.11  2.795.07  0.362.72  0.242.48  0.258.09  0.60g2 ( 24 mo)446.70  32.5625.30  3.2177.66  38.57.13  1.253.73  0.693.55  0.5112.58  1.71*means with different superscript letters are significantly different ( p<0.05 ) 
 according to analysis of variance and the student 's t test ( 5% ) . bw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular 
 circumference .. although the sc and body weight differed significantly between the two age 
 groups analyzed , there was an increase in the sc with increasing body weight . additional 
 testicular biometric parameters are often assessed in domestic animals , particularly those 
 animals used for livestock production because testicular parameters are an important part 
 of the andrological evaluation . among the available biometrics for testes , sc is one of 
 the most commonly employed because it is easy to measure and highly correlated with body 
 weight . * means with different superscript letters are significantly different ( p<0.05 ) 
 according to analysis of variance and the student 's t test ( 5% ) . bw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular 
 circumference . as shown in table 2table 2 . correlations between age , body weight and testicular parameters in murrah 
 buffalo bulls at 18 and 24 months of age  agebwsctwtltwdtttcage0.975 * 0.854 * 0.776 * 0.808 * 0.777 * 0.847 * 0.904*bw0.843 * 0.851 * 0.827 * 0.775 * 0.838 * 0.904*sc0.5170.963 * 0.932 * 0.898 * 0.931*tw0.4950.3790.4790.572tl0.982 * 0.949 * 0.963*twd0.971 * 0.965*tt0.978*tcbw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular circumference . 
 * significant ( p<0.05 ) . , there was a significant correlation between sc , age and body weight . this 
 result has been described previously in river , 
 swamp   and murrah   buffalo . bw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular circumference . 
 * significant ( p<0.05 ) . the data presented here differ from the findings in cattle , for which the sc is particularly important in identifying bulls 
 with high reproductive capacity . neither scrotal circumference nor body weight should be 
 used alone as a routine and reliable parameter to predict the reproductive capacity in 
 buffalo . the mean values and standard deviation for tw , tl , twd , tt and tc , relative to buffalo 
 age , are shown in table 1 . although the 
 average tw did not differ between the two groups , significant differences were observed 
 for tl , twd , tt , and tc ( p<0.05 ) . furthermore , these variables exhibited high 
 correlation coefficients , as noted in table 2 . body weight , age , sc , tw , tl , twd , tt and tc were positively correlated ( p<0.05 ) . this finding differs from observations 
 made in piau pigs , rams and cattle , in which a high correlation was described for all 
 parameters . the strength of the correlations obtained suggests that tl , twd , tc and tt are 
 useful parameters for the selection of breeding animals . under the husbandry conditions described here , both body and testicular development of 
 buffalo occurred more slowly than the typical growth rates for bos taurus 
 . the data for bos indicus 
 , however , suggest a similar rate of body and 
 testicular development to the rates observed for buffalo in the present study . these 
 similarities can be attributed to the fact that zebu cattle are also raised under 
 extensive farming conditions , and therefore suffer from the same nutritional consequences , 
 particularly dietary protein deficiencies , which can lead to developmental delays . in conclusion , our analysis of biometric testicular parameters in murrah buffalo revealed 
 that the tl , twd , tc and tt are useful indicators for the selection of bulls for 
 breeding . at 18 months , the testicular parenchyma contained testicular cords and internal cord 
 compartments associated with the presence of leydig cells , vessels and nerves . within the 
 testicular cords , gonocytes , undifferentiated supporting cells and luminal processes were 
 observed . gonocytes and spermatogonia in the differentiation process were identified near 
 the basal membrane ( fig . 1 . testis of murrah buffalo bulls at 18 months of age characterized by an immature 
 sexual phase ( a and b ) . we observed solid testicular cords with gonocytes ( a ) and 
 undifferentiated supporting cells ( s ) . although spermatids were present in high numbers at 18 months in nili - ravi 
 buffalo testes , spermatozoa were not present 
 in the tubule lumens analyzed here . testis of murrah buffalo bulls at 18 months of age characterized by an immature 
 sexual phase ( a and b ) . we observed solid testicular cords with gonocytes ( a ) and 
 undifferentiated supporting cells ( s ) . the testicular parenchyma at 24 months contained seminiferous tubules with lumens and 
 germinative epithelia present at different stages of the seminiferous epithelial cycle 
 ( fig . 2 . different cell types in the seminiferous epithelium of the murrah buffalo at 24 
 months of age . photomicrographs of cross sections of the seminiferous tubules 
 represent the stages of the cycle of the seminiferous epithelium . stage one ( a ) , two 
 ( b ) , three ( c ) , four ( d ) , five ( e ) , six ( f ) , seven ( g ) and eight ( h ) represent the 
 following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno ( pl ) 
 stage , primary spermatocytes at the pachytene ( p ) stage , round spermatids ( ar ) , 
 elongated spermatids ( al ) , primary spermatocytes at the zygotene ( z ) stage , primary 
 spermatocytes at the diplotene ( d ) stage , residual bodies ( cr ) and spermatozoa ( e ) . all the stages presented spermatogonia type a ( a ) and sertoli cells ( s ) . 
 hematoxylin - eosin staining . ) . the following cell types were identified : sertoli cells , spermatogonia , 
 pre - leptotene / leptotene spermatocytes , pachytene spermatocytes , zygotene spermatocytes , 
 diplotene spermatocytes , round spermatids and luminal spermatozoa ( fig . these cells could be classified into eight stages of 
 cellular associations according to the tubular morphology method . bongso   noted that the 
 spermatozoa in the tubular lumen of swamp buffalo had the same morphology at 16 months . in 
 crossbred buffalo ( mediterranean  jaffarabadi ) , this morphology was observed at 13 months 
 . ohashi   showed the onset of the spermatogenic process in buffalo at 9 months . at 24 
 months , our histological analysis of the spermatogenic process indicated 
 that these animals were not sexually mature .   different cell types in the seminiferous epithelium of the murrah buffalo at 24 
 months of age . photomicrographs of cross sections of the seminiferous tubules 
 represent the stages of the cycle of the seminiferous epithelium . stage one ( a ) , two 
 ( b ) , three ( c ) , four ( d ) , five ( e ) , six ( f ) , seven ( g ) and eight ( h ) represent the 
 following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno ( pl ) 
 stage , primary spermatocytes at the pachytene ( p ) stage , round spermatids ( ar ) , 
 elongated spermatids ( al ) , primary spermatocytes at the zygotene ( z ) stage , primary 
 spermatocytes at the diplotene ( d ) stage , residual bodies ( cr ) and spermatozoa ( e ) . all the stages presented spermatogonia type a ( a ) and sertoli cells ( s ) . 
 hematoxylin - eosin staining . mean * body and testicular biometric parameters in murrah buffalo bulls at 18 
 and 24 months of ageage groupbw ( kg)sc ( cm)tw ( cm)tl ( cm)twd ( cm)tt ( cm)tc ( cm)g1 ( 18 mo)270.25  14.5918.75  1.521.11  2.795.07  0.362.72  0.242.48  0.258.09  0.60g2 ( 24 mo)446.70  32.5625.30  3.2177.66  38.57.13  1.253.73  0.693.55 0.5112.58  1.71*means with different superscript letters are significantly different ( p<0.05 ) 
 according to analysis of variance and the student 's t test ( 5% ) . bw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular 
 circumference .. although the sc and body weight differed significantly between the two age 
 groups analyzed , there was an increase in the sc with increasing body weight . additional 
 testicular biometric parameters are often assessed in domestic animals , particularly those 
 animals used for livestock production because testicular parameters are an important part 
 of the andrological evaluation . among the available biometrics for testes , sc is one of 
 the most commonly employed because it is easy to measure and highly correlated with body 
 weight . * means with different superscript letters are significantly different ( p<0.05 ) 
 according to analysis of variance and the student 's t test ( 5% ) . bw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular 
 circumference . as shown in table 2table 2 . correlations between age , body weight and testicular parameters in murrah 
 buffalo bulls at 18 and 24 months of age  agebwsctwtltwdtttcage0.975 * 0.854 * 0.776 * 0.808 * 0.777 * 0.847 * 0.904*bw0.843 * 0.851 * 0.827 * 0.775 * 0.838 * 0.904*sc0.5170.963 * 0.932 * 0.898 * 0.931*tw0.4950.3790.4790.572tl0.982 * 0.949 * 0.963*twd0.971 * 0.965*tt0.978*tcbw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular circumference . 
 * significant ( p<0.05 ) . , there was a significant correlation between sc , age and body weight . this 
 result has been described previously in river , 
 swamp   and murrah   buffalo . bw = body weight , sc = scrotal circumference , tw = testicular weight , tl = testicular 
 length , twd = testicular width , tt = testicular thickness , tc = testicular circumference . 
 * significant ( p<0.05 ) . the data presented here differ from the findings in cattle , for which the sc is particularly important in identifying bulls 
 with high reproductive capacity . neither scrotal circumference nor body weight should be 
 used alone as a routine and reliable parameter to predict the reproductive capacity in 
 buffalo . the mean values and standard deviation for tw , tl , twd , tt and tc , relative to buffalo 
 age , are shown in table 1 . although the 
 average tw did not differ between the two groups , significant differences were observed 
 for tl , twd , tt , and tc ( p<0.05 ) . furthermore , these variables exhibited high 
 correlation coefficients , as noted in table 2 . body weight , age , sc , tw , tl , twd , tt and tc were positively correlated ( p<0.05 ) . this finding differs from observations 
 made in piau pigs , rams and cattle , in which a high correlation was described for all 
 parameters . the strength of the correlations obtained suggests that tl , twd , tc and tt are 
 useful parameters for the selection of breeding animals . under the husbandry conditions described here , both body and testicular development of 
 buffalo occurred more slowly than the typical growth rates for bos taurus 
 . the data for bos indicus 
 , however , suggest a similar rate of body and 
 testicular development to the rates observed for buffalo in the present study . these 
 similarities can be attributed to the fact that zebu cattle are also raised under 
 extensive farming conditions , and therefore suffer from the same nutritional consequences , 
 particularly dietary protein deficiencies , which can lead to developmental delays . in conclusion , our analysis of biometric testicular parameters in murrah buffalo revealed 
 that the tl , twd , tc and tt are useful indicators for the selection of bulls for 
 breeding .