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 .
 abstract buffalo are an economically important source for meat and milk production , especially in 
 brazil . however 
 , important aspects of their biology remain unknown thus far . 
 herein , we 
 describe the reproductive characteristics of male murrah buffalo ( bubalus 
 bubalis ) raised under extensive management conditions by applying biometrics 
 associated with testicular weight . 
 we analyzed seven males , divided into two groups : g1 , 
 which consisted of four 18-month - old animals , and g2 , which consisted of three 
 24-month - old animals . 
 testicular development occurs slowly in murrah buffalo , suggesting a 
 delay of sexual maturity . 
 the biometric testicular parameters analyzed were scrotal 
 circumference , testicular weight , testicular length , testicular width , testicular 
 thickness and testicular circumference . 
 our data indicate strong correlations between sc , 
 age and body weight , and additional significant relationships were identified between body 
 weight , age and other testicular parameters . 
 thus , these parameters are suitable 
 indicators when selecting bulls for breeding purposes . 
Materials and Methods
Animals
Testicular parameters
Histological processing
Statistical analysis
Results and Discussion
Histological analyses
Testicular parameters