Animals and experimental design

All the experimental procedures were performed according to the “International Guiding Principles for Biomedical Research Involving Animals” (available at: http://www.cioms.ch/images/stories/CIOMS/IGP2012.pdf). The study was conducted in the state of Queretaro, Mexico (20º 43’ N, 100º 15’ W). Forty-three summerborn (July to September) Barbados Blackbelly ram lambs were used. Lambs were always housed in outdoor pens; before weaning, lambs were creep fed with a diet containing 14 % crude protein (78 % sorghum, 20 % canola meal and 2 % minerals); after weaning at 75 d of age, lambs were offered alfalfa hay and a concentrate as described above, water was always available ad libitum. Lambs were allotted into eight experimental groups, each group were surgically castrated under local anesthesia either, at birth (before 3 d of age), or when they were 3, 6, 9, 12, 15, 18 or 21 wk of age (n= 4 to 6 per group), within each group the difference in age was never greater than 3 d. All castrations were performed during September to December.

Tissue collection and processing

Both epididymides were trimmed off extraneous tissue and weighed separately. The left epididymis was divided in its three anatomical regions, caput, corpus and cauda⁵. Each region was weighed and the percentage accounted for each region to the whole organ weight was calculated, these percentages were then used to estimate the paired weight of each region. Samples were obtained from the left epididymis, from the zones 2 (caput), 5 (corpus) and 8/9 (cauda)(5), and were fixed and processed for histology¹⁰. Three to four sections (5 μm) were made from each sample, mounted onto gelatin-coated glass slides and dried overnight at 37 °C. Sections were stained with hematoxylin-eosin and stored at room temperature until quantitative morphometric analysis was conducted.

Morphometric evaluation of epididymal sections

One or two sections from each epididymal region and ram were randomly chosen and observed under a bright field microscope (Axiostar Plus, Carl Zeiss, Mexico DF); digital images were obtained at 100x or 200x magnifications (depending on the age of the lamb and the variable to be recorded) with a digital camera (Color View II, Soft Imaging System, Lakewood CO) attached to the microscope. Image acquiring and processing were performed using image analysis software (AnalySIS Opti Basic, Soft Imaging System). The percentage of the epididymal parenchyma composed of tubular tissue in each region was determined in images taken at a magnification of 100x. Each section was divided in quadrants, a field from each quadrant was randomly chosen and a square (945.5 x 945.5 μm) was applied; then, all tube sections inside the square were delimited and the software calculated the total tubular area inside the square, which was used to calculate the corresponding percentages. Tubular variables of the epididymal tissue were obtained by tracing outlines of 10 rounded tube sections in every region for each ram lamb, including and not including the muscular layer, also by tracing the tubular lumen. The analysis software calculated several variables, such as tubular area, perimeter and diameter. Such data were used to calculate the tubular diameter, the thickness and area of the muscular layer, the height and area of the epithelium, and the diameter and area of the tubular lumen in every epididymal region in each lamb.

Statistical Analyses

The 10 values for each epididymal region within a lamb were averaged; in this way a single value for a particular epididymal region and lamb was obtained for the statistical analysis. Data were analyzed as a completely randomized design with a factorial arrangement, using the GLM procedure of SAS (SAS Institute Inc., Cary NC, USA). The age of the animals, the anatomical region and the interaction age by region were included in the model; the PDIFF option of SAS was used to compare least square means among age groups and among regions. In order to meet the assumptions of the analysis of variance, percentage data, expressed as proportions (p), were transformed to arc-sine squared root of p; all the other variables were log_n transformed. After analysis, data were transformed back to actual values for tables and figures.

Epididymal weight

Differences between ages for paired epididymis weight were always significant (P<0.05), except for 0 vs 3, and 18 vs 21 wk. At birth, the weight of paired epididymides was 0.93 ± 0.07 g (Figure 1), during the first 9 wk increased 5.4 fold (5 ± 0.49 g); the greatest growth rate was observed between 9 and 18 wk (4.7 fold), until reaching 25.6 ± 2.5 g on wk 21. The interaction age x region was significant (P<0.001); paired weight of each epididymal region followed a similar trend, yet with some differences among regions. Corpus epididymidis was always lighter than cauda (P<0.01), and lighter than caput from wk 6 onwards (P<0.01), from 0.14 ± 0.01 g at birth to 4.6 ± 0.31 g at wk 21. At birth, the weight of caput (0.34 ± 0.03 g) and cauda (0.44 ± 0.03 g) were not different to each other, as well as by 3 wk. Between 6 and 12 wk, the cauda weight increased faster and was greater than caput (P<0.01). After wk 12, the caput weight started increasing, in such a way that by 15 to 21 wk no differences were detected between the two regions (caput 10.7 ± 1.03 g; cauda 10.3 ± 0.89 g at wk 21).

Each time point represent the mean ± SE for 4 to 6 rams. *Corpus weight was smaller than caput from 6 wk onwards (P<0.01), and smaller than cauda at every age evaluated (P<0.01). **Cauda weight was larger than caput at 6, 9 and 12 wk of age (P<0.01). The arrow indicates the age when sperm first appeared in the tubular lumen of the three epididymal regions.

Figure 1 Development of paired epididymal weight and paired weight of each anatomical region, from birth to 21 wk of age in Barbados Blackbelly ram lambs. 

The differential development of the epididymal regions was also evident on the percentage that each region accounted to the total organ weight (interaction age x region, P<0.001; Figure 2). The proportion accounted by the caput decreased between birth (36.7 %) and wk 9 (28 %; P<0.001), then increased until wk 15 (P<0.05), with no further changes thereafter until wk 21 (41.7 %). The cauda followed an opposite trend, increasing from birth (47.8 %) to wk 6 (58.5 %; P<0.001), decreasing from wk 9 to 15 (P<0.001), with no further changes thereafter until wk 21 (40.3 %). Cauda was greater than the other regions from birth to wk 12 (P<0.001), and not different from caput at wk 15, 18 and 21; corpus accounted always the smallest percentage (P<0.001).

Each time point represents the mean ± SE for 4 to 6 rams. *Cauda was greater than caput from 0 to 12 wk of age (P<0.001); corpus always accounted for the smaller percentage to the total epididymal weight (P<0.001). The arrow indicates the age when sperm first appeared in the tubular lumen of the three epididymal regions.

Figure 2 Development of the percentage accounted by each epididymal region to the total epididymal weight, from birth to 21 wk of age in Barbados Blackbelly ram lambs. 

Characteristics of the epididymal tissue

Tubular tissue percentage and tubular diameter. The interaction age by region was significant (P<0.05) for the percentage of tubular tissue; this variable had an increasing trend in the three epididymal regions until wk 15 (Table 1); then, it did not change (caput and corpus) or decreased (cauda) by wk 21. Overall, the percentage of tubular tissue was greatest in the cauda and lowest in the caput (P<0.05), except for wk 3, 18 and 21, when no differences among regions were detected; corpus was intermediate, but not always different from the other regions. The increasing rate (percentage of each age, related to wk 21) was different among regions (Figure 3a); by wk 6, corpus and cauda had reached, respectively, 93 and 88 % of the final value, whilst caput had reached only 68 %, such trend was maintained until wk 15. For tubular diameter, the interaction age by region was not significant (P=0.13; Table 1). This variable increased (P<0.05) until wk 15 (caput and corpus) or 12 (cauda), with no changes thereafter. The greatest diameter was always observed in the cauda, and the smallest in the caput (P<005); corpus was not different from caput, except for wk 3 and 6, when all regions were different to each other. Regarding the increasing rate (Figure 3b), by wk 6, caput, corpus and cauda had reached 38, 47 and 55 % of their final values, respectively; similar differences were maintained until wk 12, at that age cauda had reached 92 %, whilst similar percentages (90 %) were not reached in caput and corpus until 18 wk of age.

Table 1 Mean (± SE) of the percentage of tubular tissue and the average tubular diameter in the several epididymal anatomical regions in Barbados Blackbelly ram lambs, from birth to 21 wk of age. 

^a-e Means within a column without a common superscript are different (P<0.05).

^x,y,z Means within a row and variable without a common superscript are different (P<0.05).

Data are the percentage of the values at each age as compared with the value at 21 wk of age for each variable (panels a, b, d). * For epithelium height (panel c), the percentage value at each age is relative to the maximum value reached in each region (21 wk for caput and corpus, and 9 wk for cauda).

Figure 3 Relative increase of the epididymal tubular tissue percentage (panel a), tubular diameter (panel b), epithelium height (panel c) and epithelium area (panel d) in Barbados Blackbelly ram lambs. 

Epididymal muscular layer. The interaction age by region was significant (P<0.01) for both variables of the muscular layer. The thickness of the muscular layer in caput did not change between birth and wk 6 (Table 2), increased 35 % by wk 9 (P<0.05) and then, did not change or had a descendent trend until wk 21. In the cauda, the muscular thickness did not change between birth and wk 6, increased 119 % by wk 9 (P<0.05), with no change by wk 15 and a descendent trend thereafter (P<0.05). In the corpus epididymidis there were no changes between 3 and 21 wk. When comparing the muscular layer thickness in wk 0 vs wk 21, no differences were observed in the caput and corpus, but in the cauda, it was 48 % greater on wk 21 (P<0.05). The muscular layer area (Table 2) increased until wk 12 (caput and corpus) and 9 (cauda), with no significant changes thereafter. The maximum area observed in each region, as compared to value at birth, was 3.8-, 3.8-, and 5.5-fold, for caput (wk 18), corpus (wk 18) and cauda (wk 15), respectively. Comparisons among regions were similar for both variables, the greatest values were observed in the cauda (P<0.05), followed by the corpus and caput; however, differences between the last two were significant (P<0.05) only at 3 and 6 wk.

Table 2 Mean (± SE) of the thickness and area of the tubular muscular layer in the several epididymal anatomical regions in Barbados Blackbelly ram lambs, from birth to 21 wk of age. 

^a-e Means within a column without a common superscript are different (P<0.05).

^x,y,z Means within a row and variable without a common superscript are different (P<0.05).

Epididymal epithelium. The interaction age by region was significant (P<0.001) for both variables of the epithelial layer. In the caput and corpus, the epithelium height did not change between birth and wk 6, then increased until wk 12 (caput) or 15 (corpus), with no further changes (Table 3); in the cauda, an increasing trend was observed from birth to wk 9, then, decreased on wk 12, with no more changes until wk 21. Comparison between regions had two different phases, from birth to wk 9, the epithelium was highest in the cauda and lowest in the caput (P<0.05), but from 12 to 21 wk the situation reversed, as the lowest values were observed in the cauda (P<0.05) and the greatest in the caput and corpus with no differences between the last two. Regarding the increasing rate (Figure 3c), at birth, cauda had over 45 % of their maximum value, which was reached at wk 9; caput and corpus, at birth had reached, respectively, 20 and 25 % of the value at wk 21, and did not reach over 90 % until wk 15. The epithelial area (Table 3) did not change between birth and wk 6 (caput and corpus) or 3 (cauda), then, it increased until wk 15 in the three regions (P<0.05), with no further changes (Table 3). Between birth and wk 12, the greatest values were observed in the cauda, with the smallest values in the caput (P<0.05); from wk 15 onwards no differences among regions were detected. The increasing rate was always greater for cauda (Figure 3d), by wk 6 it had reached 31 %, as compared with 5 and 8 % for caput and corpus, respectively; such differences were maintained until wk 18.

Table 3 Mean (± SE) of the tubular epithelium height and area in the several epididymal anatomical regions in Barbados Blackbelly ram lambs, from birth to 21 wk of age. 

^a-d Means within a column without a common superscript are different (P<0.05).

^x,y,z Means within a row and variable without a common superscript are different (P<0.05).

Epididymal tubular lumen. The interaction age by region was not significant (P≥0.25) for both variables of the tubular lumen, but the effect of region was (P<0.001). The lumen diameter and area did not increase or increased very little in all three regions until wk 6; then, increased steadily until wk 15 (caput and corpus) or 12 (cauda), with no further changes thereafter (Table 4). The greatest relative augment was observed between 9 and 12 wk (0.5- to 1.2-fold in the lumen diameter, and 1.3- to 5.2-fold in the lumen area), with the greatest increase in the cauda; both variables were greatest in the cauda at all ages (P<0.05), with no differences between caput and corpus. The maximum lumen diameter observed in each region (caput, corpus and cauda, respectively) corresponded to 3.7-, 3.5- and 5.2-fold increase, as compared to wk 0. Similarly, the increase in the lumen area represented 21.8-, 21.2- and 24.9-fold, as compared to wk 0, for caput, corpus and cauda, respectively. Sperm first appeared in the tubular lumen of the three epididymal regions by wk 18 (arrows in Figures 1 and 2), which indicate that by that age the lambs had already reached or were very close to reach puberty.

Table 4 Mean (± SE) of the tubular lumen diameter and area in the several epididymal anatomical regions in Barbados Blackbelly ram lambs, from birth to 21 wk of age. 

^a-d Means within a column without a common superscript are different (P<0.05).

^y,z Means within a row and variable without a common superscript are different (P<0.05).