Treatments, animals and management

Fifteen (15) male pubertal Pelibuey sheep (aged 6 ± 0.5 mo) with an average weight of 24 kg ± 3.95 were utilized and distributed in a fully random way in three treatments (n=5): T1= integral diet with 40 % M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.). T2= integral diet with 40% Trichanthera gigantea + Taiwan grass. T3= integral diet + Taiwan grass. The amount of feed offered was 4.7 % of the live weight during the 90 days of the experiment (the consumption was adjusted every seven days). Table 1 shows the contents of the percentage composition of the experimental diets, and Table 2, the proximate analysis.

Development of lambs

To determine the development of lambs, the animals were weighed every seven days; prior to each weighing, the animals underwent a period of 18 h of fasting. They were weighed on a T31P (OHAUS) electronic platform scale with a capacity for 300 kg, with a margin of error of 0.1 kg. The daily weight gain was determined through the analysis of these weights¹⁶.

Testicular development

The measurement of the scrotal circumference was taken with a plastic measuring tape, while the testicular diameter was registered using a TRUPER brand digital caliper ¹⁷.

Semen quality

For the assessment of the semen quality, after the 70-days experimental period (at age 8 ± 0.7 mo), six semen samples per animal (two per week) were collected in each treatment using an estrogenized female dummy and the artificial vagina method with a rubber sleeve (Liner). Warm water at 45 ⁰C and air were introduced by blowing through a valve in order to obtain the temperature and pressure required to stimulate ejaculation¹⁸. Once the ejaculates were obtained, they were transported to the laboratory in isothermal conditions (35 to 37 ⁰C) for evaluation. At the laboratory, the samples were placed in a double boiler at 37 ⁰C for immediate evaluation.

The following were determined in the collected samples:

1) The volume of the ejaculate, with a graduated pickup tube.

2) The sperm concentration was determined with the CASA system (ISAS®v1 (Proiser R&D, Valencia, Spain) by diluting 5 µl of pure semen in 995 µl of distilled water (1:200); 9 µl of the diluted sample were then placed in a Bürker chamber for analysis. Sperm motility was evaluated by depositing 5 µl of diluted sample at a concentration of 30 x 10⁶ spermatozoa/ml on a Makler® counting chamber (Sefi Medical Instruments, Haifa, Israel), and four recordings were made of each sample; the dilution was performed with the commercial diluent (Triladyl ® + distilled water + 20 % egg yolk). The parameters evaluated for this variable were: percentage of total motility (TM), percentage of progressive motility (PM), curvilinear speed (CLS, µm/s), rectilinear speed (RLS, µm/s), average speed (AS, µm/s), linearity index (LIN), straightness index (STR), oscillation index (WOB), average amplitude of lateral displacement of the head (ALH), sperm head beat frequency (HBF).

3) The sperm viability was assessed by staining with the Fluorochrome SYBR-14/IP (Live/Dead® kit L-7011, Invitrogen^TM); 1 µL of SYBR-14 (10 µM) was mixed with 1 µl IP (12 µM) in 100 µl of diluted sperm sample and kept for 10 min at 37 ⁰C; 5 µl of the stained sample were subsequently placed on a glass slide preheated at 37 ⁰C for evaluation in a (CX-31, Olympus, Tokyo, Japan) fluorescence microscope with a wavelength of 488 nm.

4) The mitochondrial activity was determined using the J-C1 fluorochrome (153 µM, Molecular Probes® T-3168, InvitrogenTM).1 µl of the fluorochrome was mixed with 100 µl of diluted sperm sample and kept for 10 min at 37 ⁰C; 5 µl of the stained sample were subsequently placed on a glass slide preheated to 37 ⁰C for evaluation under a fluorescence microscope with a wavelength of 488 nm.

5) The integrity of the acrosomes was evaluated with the FITC-PSA fluorochrome (100µg/mL, L-0770, Sigma-AldrichTM), adding 5 µl of the fluorophore in 100 µl of diluted sperm sample and was kept for 30 min at 37 ⁰C; 5 µl of the incubated sample were subsequently placed on a slide and were evaluated in a fluorescence microscope with a wavelength of 485 nm.

The data obtained were analyzed using the PROC GLM procedure of the statistical package SAS 9.4 for Windows (Inst. Inc. Cary, NC, USA); a repeated means variance analysis (ANOVA) a Tukey mean comparison test were performed in order to evaluate the effect of diet on the sperm quality.

Productive behavior

The daily weight gains of the studied animals did not exhibit any differences (P˂0.05) between treatments. These gains are greater than those recorded by Madera-Solis et al¹⁹, who with different levels of inclusion of Leucaena leucocephala (Lam.) de Wit found daily weight gains of 25, 41.67, 50, and 75 g in Pelibuey sheep. For their part, Rivers et al²⁰ reported profits of 54, 87 and 56 g/d when assessing the use of Morus sp. and Gliricidia sepium Kunth ex Walp. as substitutes for concentrated feed for growing lambs. Daily revenues of 45.5, 96.3, 124.5 and 106.4 g with different levels of inclusion of hay from Hibiscus rosa-sinensis L. are also reported in growing sheep⁴. The profits obtained in this study are less than those mentioned by others¹⁶, which recorded daily gains of 229 ± 10 g/d for Pelibuey-Katadin animals housed in elevated cages and fed a commercial concentrate with a 16 % crude protein content and Cynodon nlemfuensis Vanderyst hay.

When assessing the performance of the carcass, no differences were found (P˂0.05) between treatments: 50.79, 49.44 and 46.94 % for treatments T1, T2 and T3, respectively; these values are similar to those registered by Magaña-Monforte et al¹⁶, who obtained a yield of 49.1 ± 0.58 with crossbred animals of the Pelibuey-Katahdin breeds. The yields of this study were lower than those found in another research²¹, which cites a carcass output of 52.8 % for whole males, 56.64 % for castrated males, and 55.87 % for females. The proper nutritional balance present in the assessed diets generated an adequate, similar productive response in the study animals, compared to other researchers performed under similar conditions. These results in the evaluated rations (Table 2) can be attributed to the adequate level of protein and the improved nutritional balance²²; this stimulates an increase in the level of efficiency of nutrients produced by greater microbial activity in the rumen²³ and ensuring metabolic stability²⁴, positively affecting the various productive parameters evaluated in the present study (daily weight gains and carcass output).

Testicular development

Testicular volume is an indicator of the fertilizing ability of the male and of the highest fertility; also a smaller number of abnormal spermatozoa has been observed in animals with a larger scrotal circumference. In the study animals, the testicular development determined by means of the increase in scrotal circumference and diameter exhibited differences (P<0.05) between the treatments evaluated for both variables (Table 3). For the variable scrotal circumference, treatment T3 had a higher mean than T2, while T1 did not differ (p>0.05) from the other two treatments. For the scrotal diameter variable, there was no difference between treatments T1 and T3; however, both were larger than with T2.

The means for the scrotal circumference were: 26.4 ± 0.33, 25.4 ± 0.33 and 26.6 ± 0.33 cm for treatments one two and three, respectively (Table 3); they are lower than those recorded by Luna et al.²⁵, who found averages of 28.4 cm in sheep supplemented with animal fat and palm oil; as well as those reported in other studies^26,27, of 29.4 and 36.5 cm, respectively. The averages of scrotal diameter were lower than those recorded by Ballin et al.²⁶. Testicular development is determined by various factors, mainly age, race, and nutrition²⁷; in this regard the study animals, with an average age of 7±0.5 months, received a diet with adequate levels of protein and nutrients required for meeting the nutritional requirements according to their physiological stage, which is reflected in the weight gain and in the obtained testicular development.

Seminal evaluation

The variables of the ejaculate volume and sperm concentration did not exhibit any differences (P>0.05) between treatments (Table 3). The volume for the three treatments lies within the range considered normal, between 0.5 and 2.0 ml, and this volume varies, depending on the age, size, body condition of the animal, frequency of collection, and dexterity of the operator²⁸. Despite this, the values reported in this experiment were lower than those reported by Mansano et al²⁹, who obtained volumes of 1.3 ± 0.4 ml; Carrillo and Hernández³⁰ obtained 1.41 ± 0.11 ml; for their part Córdova-Izquierdo et al³¹ obtained 1.11 ml; however, these two studies were performed with mature males unlike the pubertal males (7 ± 0.5 mo of age) used in the present study.

The low ejaculate volume values obtained in this study, contain a greater concentration of spermatozoa per ml. Therefore, these values are higher than those recorded in other studies, which indicate ranges of 206.4 to 711.89 × 10^6(30,31, but similar to those found by Domínguez et al³², of 2.19 ± 0.04 and 108 ± 8.0 × 10⁶ spermatozoa/ml, when assessing the semen quality of F1 Katahdin × Pelibuey lambs supplemented with alfalfa. In contrast, the concentration values obtained here are lower than those found by Quintero et al³³, who obtained 2,628.2 × 10⁶ when assessing hair sheep aged 2 to 5 yr

There were no differences (P<0.05), in total motility, curvaceous speed, and AD, where treatments T1 and T2 were better than T3; and in the variables AS and RLS, T1 exhibited the best results with respect to T3; however, no differences were observed between T1 and T2. For the rest of the analyzed variables (PM, LIN, IR, OI, and BF), no differences were found between experimental groups (Table 4).

The results for the motility variable are higher than those recorded by Mansano et al²⁹, of 52 %, but similar to those found in another research³², which registered an 80 % total motility and over 20 % progressive motility; however, the results of this study are lower than those recorded by Quintero et al³³, who obtained 90.69 % progressive motility.

In the analysis of the plasma membrane viability variable, differences (P<0.05) were found between the assessed treatments (T1: 54 %, T2: 63 % and T3: 21 %), T2 being the treatment that exhibited the highest percentage of viability, in contrast with T3, which obtained the lowest percentages (Figure 1).

T1: integral diet with 30% M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.), T2: integral diet with 30% T. gigantea + Taiwan grass, and T3: commercial feed + Taiwan grass.

Figure 1 Feasibility, mitochondrial activity and integrity of the acrosome of hair sheep supplemented with Moringa oleifera and Trichanthera gigantea 

The mitochondrial activity and acrosome integrity variables did not reveal any differences (P>0.05) between treatments, while T1 exhibited the highest means for the mitochondrial activity variable, T3 showed the highest percentages for the mitochondrial activity variable (Figure 1). The integrity of the plasma and acrosome membranes and mitochondrial activity are major factors for fertilization, as an intact plasma membrane indicates that the spermatozoon is alive; an intact acrosomal membrane allows penetration of the oocyte, and the mitochondrial activity ensures the presence of energy required to move the spermatozoon (Figure 2). For this reason, it is essential to include it in the assessments on semen quality³¹. The results observed in this study agree with those recorded by Cordova-Izquierdo et al³¹ who obtained 68.5 % of the integrity of the plasma membrane by assessing sheep of the Suffolk race. They also match the results of Rubio-Guillen et al³⁴, who recorded 42 % when evaluating West African sheep. However, the percentages obtained in this study are lower than those found in other researches^30,32,33 but are consistent with the values estimated by Domínguez et al³² for acrosomal integrity.

A. Living (green) and dead (red or orange) sperm heads stained with SYBER-14/IP fluorochromes; viability of the acrosome located in the apical region using the fluorochrome FITC-PSA (colorless: viable; green: corrupted). B. Mitochondrial activity located in the middle region of the tail stained with the fluorophore JC1, with activity (yellow or orange) and without activity (green).

Figure 2 Sperm cells from sheep fed with Trichanthera gigantea or Moringa oleifera 

The parameters recorded in the assessment of the semen quality as well as of the testicular development may be attributed to the adequate nutritional balance of the assessed diets, as sperm production and quality, and testicular size, are directly influenced by nutrition. An increase in the nutritional level increases the frequency of pulses of LH and increases levels of FSH³⁵, hormones that promote spermatogenesis and androgen secretion³⁶.

The animals assessed in the present study are pubescent, animals, and animals in this condition have a greater amount of immature, dead or defective spermatozoa, which in turn produce high amounts of free radicals that affect those spermatozoa that are in good conditions, since free radicals cause a cascade of lipid peroxidation affecting mainly the unsaturated fatty acids of the plasma membrane of the sperm, causing loss of sperm function and cell apoptosis, and therefore are one of the leading causes of death of sperm cells³⁷.

It has been observed that the inclusion of antioxidants in the diet improves the percentage of live sperm cells because antioxidants eliminate the free radicals that are found in the seminal plasma and confer protection to the spermatozoa^13,14; Priyadarshani and Varma³⁸ observed a higher percentage of live cells when they used M. oleifera in the diet of mice. This result is similar to what has been observed in the present study and may be ascribed to the contribution of antioxidants (beta-carotene) contained in M. oleifera and T. gigantea¹⁵.

The secondary compounds present in both assessed species may be related to the values found in the present study; therefore, further studies determining the presence and concentration of these would contribute greater evidence in this regard.