Feed intake, sperm output and seminal
characteristics of Ethiopian highland sheep
supplemented with different levels of leucaena
(
Leucaena leucocephala
) leaf hay
Negussie Dana
a,*, Azage Tegegne
b, Teshome Shenkoru
a aDebre Zeit Agricultural Research Center, P.O. Box 32, Debre Zeit, Ethiopia bInternational Livestock Research Institute, P.O. Box 5689, Addis Ababa, Ethiopia Received 7 September 1999; received in revised form 15 February 2000; accepted 2 May 2000Abstract
The effect of feeding increasing levels of Leucaena leucocephala leaf hay on the potential fertility and feed intake of male highland sheep maintained on roughage diet was studied. In treatment 1 (NS), animals received only chickpea haulm. Treatments 2 (L100), 3 (L200) and 4 (L300) consisted of 100, 200 and 300 g per head per day of sun dried leucaena leaf hay while sheep under treatment 5 (C300) received 300 g per head per day of concentrate supplement. Chickpea haulm was offered ad-libitum to all groups and water and mineral licks were freely available.
Addition of leucaena improved total dry matter (DM) and crude protein (CP) intake signi®cantly without depressing the intake of the basal diet. Supplementation increased the percentage of motile cells (10 vs. 76% for L300 ) and mass activity/motility score (1.5 vs. 3.2 for L300) of spermatozoa while reducing the incidence of total morphologically defective sperm cells (34 vs. 5% for L200). Volume of ejaculate (0.36 vs. 1.1 ml for L200), sperm concentration (2.8 vs. 7.1109mlÿ1
for L300), and total number of spermatozoa per ejaculate (1.96 vs. 5.92109per ejaculate for L300),
increased signi®cantly as a result of supplementation. Testicular size showed signi®cant differences among treatment groups and generally increased with supplementation. It was concluded that supplementation of up to 300 g per head per day of leucaena resulted in improved feed intake, testicular growth, sperm production and semen quality of Ethiopian highland sheep offered chickpea haulm basal diet.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Highland sheep; DM intake;Leucaena leucocephala; Chickpea haulm; Seminal characteristics 86 (2000) 239±249
*Corresponding author. Tel.:251-1-338555; fax:251-1-338061.
E-mail address: dzarc@telecom.net.et (N. Dana).
1. Introduction
Forage supplements have enormous potential for ruminant production in the tropics because of their easy availability in the farms; high nutritive value and reduced feeding cost. Of the forage supplements used, legumes have been particularly advantageous.
Leucaena, a native of Central America, is a tropical legume of mimosa family. It exists through out the cool subtropics and equatorial subtropics. Of all tropical legumes it has the widest variety of uses one of which is its use as forage (NAS, 1977). Especially, the drought tolerance nature of this plant makes it a potential feed source to increase meat and milk production during the dry season.
In some countries like Ethiopia the use of leucaena as animal feed is limited mainly by its content of the toxic non-protein amino acid, mimosine (b-(N-(3-hydroxy-4-oxypyridyl))-a
aminopropionic acid) and the rumen degradation product 3-hydroxy-4 (1H)-pyridone (3,4-DHP) (Hammond, 1995). The aromatic amino acid, mimosine occurs mainly in the seeds and leaves. It is responsible for toxic effects such as abortion and infertility (Hamilton et al., 1971), inhibition of deoxyribonucleic acid (DNA) replication, organ damage and even death (D'Mello, 1992) while DHP is a potent goiterogen (Hammond, 1995).
Information on the effect of feeding leucaena on the reproductive performance of ruminants is scarce and often inconsistent. For instance, inclusion of up to 75% of leucaena in the ration resulted in no adverse effect on the reproductive performance of goats in Philippines (Abilay and Arinto, 1981). On the other hand, a review by Hammond (1995) revealed that leucaena in unadapted cattle resulted in reduced calving percentage due to early embryonic mortality. The in¯uence of feeding different levels of leucaena on sheep reproduction is not well known. Hence, this study was conducted to assess the effect of feeding increasing levels of leucaena on the feed intake and quality and output of semen of highland sheep maintained on chickpea haulm as basal diet.
2. Materials and methods
2.1. Description of the study area
The study was conducted at Debre Zeit Agricultural Research Center (DZARC) located 45 km East of Addis Ababa at an altitude of 1900 m a.s.l. Based on 22 years data, the average annual rainfall of the area is 851 mm and the average minimum and maximum temperatures range from 8.9 to 24.38C, respectively, the mean average being 16.68C. According to the weather report of DZARC (1996), annual rainfall of 950 mm, average minimum, maximum and mean temperatures of 11.5, 26.5 and 198C, respectively, and relative humidity of 56.4% were recorded during the study period.
2.2. Animals and management
Sheep were quarantined upon arrival at the station and treated against endo- and ecto-parasites. The animals were randomly divided in 10 groups housed in group pens and each group randomly allocated to one of the ®ve treatments that was replicated twice.Data on feed intake and seminal characteristics were taken after the animals were given adaptation period to the environment and experimental diets for 30±40 days. The experiment lasted for 15 weeks.
In treatment 1 (NS) animals were offered only chickpea haulm, while those on treatments 2 (L100), 3 (L200) and 4 (L300) were supplemented with 100, 200 and 300 g per head per day of sun dried leucaena leaf hay. Animals on treatment 5 (C300) were supplemented with 300 g per head per day of concentrate mixture. The concentrate was composed of wheat bran, noug (Guizotia abyssinica) cake, molasses and salt and was formulated to supply about 200 g CP/kg DM. Table 1 presents the chemical composition of feeds used in the experiment.
The basal diet, chickpea haulm, was fed ad-libitum to allow 25% feed refusal (as-fed basis) based on intake of the previous day. Mineral licks and water were freely available at all times. Chickpea haulm was offered once and the supplements twice daily (10:00 and 16:00 h).
2.3. Data and sample collection and analysis
2.3.1. Feed intake and composition
Amount of feed offered and refused was recorded daily to determine feed intake. Daily samples of feeds offered and refused were collected, bulked weekly and subsamples taken after thorough mixing. The samples were analyzed for dry matter (DM), organic matter (OM) and crude protein (CP) according to AOAC (1985). Neutral detergent ®ber (NDF) and acid detergent ®ber (ADF) were determined using the procedures of Goering and Van Soest (1970).
2.3.2. Seminal measurements
Semen was collected fortnightly by using an electro-ejaculator (probe for sheep and goats, Lane Manufacturing Inc., USA) after training the sheep for few weeks. Ejaculate volume was determined by collecting semen into graduated tubes. The percentage of motile spermatozoa (motility percent, MOP) and wave motion (motility score, scored from 1 to 5) were estimated immediately after ejaculation by microscopic examination (10) of a drop of semen placed between a slide and 2232 mm cover slip in a warm (34±378C) stage (Salamon, 1976). The wave motion (motility score) was de®ned as good when the sperm cells showed a vigorous straight forward movement and poor when a Table 1
Chemical composition of chickpea haulm,Leucaena leucocephalaleaves and concentrate mixture (% of DM)
Feed DM OM CP NDF ADF Ca P
Chickpea haulm 94 82.3 5.6 57.3 41 0.72 0.11
Leucaena 92 78.9 24.4 23.9 11.6 1.48 0.19
Concentrate 93 72 21.9 20.9 10.7 0.47 0.60
weak, slow and spasmodic movement of the sperm cells was noted (Kemp et al., 1989). Fifty microliter of semen samples were dropped into tubes containing 2.9% PBS-glutaraldehyde solution for subsequent determination of sperm concentration and evaluation of sperm morphology. Sperm cell counts were made in duplicate using Neubar haemocytometer and a phase contrast microscope (600) as described by Moss et al. (1979). The number of sperm cells produced per ejaculate was calculated for each sheep by multiplying the volume of ejaculate with the sperm cell concentration.
The percentage of morphologically abnormal spermatozoa was determined adopting the classi®cation scheme proposed by Garner and Hafez (1980). Spermatozoa were examined microscopically on unstained smear of each semen sample, and the percentage of defective sperm cells was determined from a count of 300 cells per smear.
2.4. Statistical analysis
The data were subjected to variance analysis techniques according to the general linear model (GLM) procedures of SAS (SAS, 1994). Treatment means were separated using Tukey's studentized range test protected by a signi®cantF-test for treatment effects. Data that were found to be deviating from the assumptions of the analysis of variance were transformed before running the analysis. Accordingly, the data on concentration and number of spermatozoa were log transformed, while square root transformation was applied to morphological data. Relationships among parameters were analyzed using correlation analysis.The statistical model used in the analysis was:
Yijmaieij
wherem is the overall mean,aithe treatment effect andeijthe random error term.
3. Results
3.1. Dry matter and crude protein intakes, and feed conversion ratio
Daily DM intake and animal performance are shown in Table 2. Leucaena supplementation increased signi®cantly total DM intake but did not depress intake of the basal diet. However, differences in total DM intake between levels of leucaena were not signi®cant.
The CP intake differed signi®cantly (p<0.001) among treatments. Sheep receiving 300 g per head per day of leucaena leaf hay had the highest CP intake, increasing more or less proportionately to the levels of leucaena inclusion. Animals fed on chickpea haulm alone had the lowest feed utilisation ef®ciency. But this was improved signi®cantly with supplementation (Table 2).
3.2. Seminal characteristics
morphological defects of sperm cells. Spermatozoa of rams receiving only chickpea haulm showed signi®cantly lower progressive motility, mass activity and higher morphological abnormalities (both major and minor) than the rest. The other treatment groups did not differ from each other (p>0.05) in these parameters. Supplementation Table 2
Dry matter (DM) and crude protein (CP) intakes, weight gain and feed conversion ratio (S.E.) of sheep supplemented with 100 (L100), 200 (L200) and 300 (L300) g per head per day of leucaena leaf hay and 300 g per head per day of concentrate (C300)
Parameters Treatmenta
aLeast-square means in row with different letters are signi®cantly different from each other (p<0.05). bNS: not supplemented.
Table 3
Effects of supplementing 100 (L100), 200 (L200) and 300 (L300) g per head per day of leucaena leaf hay and 300 g per head per day of concentrate (C300) to rams offered chickpea haulm on the quality and quantity (S.E.) of semen collected by electro-ejaculation
Parameters Treatmenta
aLeast-square means with the same letters in the same row are not signi®cantly different from each other (p>0.05).
bNS: not supplemented.
cAL-MOP: anti-logarithm of motility percentage (%).
dAL-CONCT: anti-logarithm of sperm cell concentration (109mlÿ1). eAL-TSPZ: anti-logarithm of total spermatozoal output (109per ejaculate). fSC: scrotal circumference (cm).
gMorph. defects: morphological defects.
signi®cantly increased the volume of ejaculates, sperm concentration and the total number of spermatozoa per ejaculate.
The quantity of morphologically abnormal spermatozoa in the ejaculates was signi®cantly reduced as a result of supplementation. The percentage of normal spermatozoa for treatments L100 to C300 ranged from 88.5 to 95%. These treatments did not differ from each other in the extent of sperm cells with major (primary) sperm cell defects while a signi®cant difference was noted in the proportion of spermatozoa having minor morphological abnormalities between L100 and L200.
3.3. Scrotal circumference
A highly signi®cant difference was observed in scrotal circumference (SC) of rams receiving the different treatment rations (Table 3). Addition of 100, 200 and 300 g per head day of leucaena in the diet increased testicular size by 20, 24 and 21%, respectively, while a 25% increment was achieved with the concentrate supplement compared to the control basal diet.
4. Discussion
4.1. Dry matter and crude protein intakes, and feed conversion ratio
The CP content of chickpea haulm was below the critical threshold of 7±8% to avoid that voluntary intake and digestibility could be affected (Van Soest, 1982). Values obtained for leucaena were comparable to those reported earlier by NAS (1977).
Inclusion of up to 300 g per head per day of leucaena in the diet did not have any signi®cant effect on the daily dry matter intake of the basal diet, although a positive effect was noted. Results on the effect of fodder legume supplementation on intake of basal diets were inconsistent. Topps (1995) suggested that supplementation of fodder legumes at levels less than 30±40% increases intake of basal diets. However, leucaena supplementation was reported to reduce the voluntary intake of hay in Tanzanian goats (Mtenga and Shoo, 1990). A study by Tomkins et al. (1991) also showed that supplementation (of about 30% of the ration) of dried L. leucocephala leaves to rams reduced intake of pangola grass basal diet by 8%.
Total daily DM intake, on the other hand, was signi®cantly improved by leucaena supplementation con®rming earlier reports by Mtenga and Shoo (1990) and Tomkins et al. (1991). This would be expected since nitrogen supplementation to ruminants increases feed intake by increasing the rate of fermentation in the rumen (Van Soest, 1982) and thereby resulting in improved digestibility and faster rates of passage of digesta through the gastro-intestinal tract (Preston and Leng, 1987).
4.2. Seminal characteristics
4.2.1. Volume of ejaculate
The minimum ejaculate volume, recorded with the control diet (chickpea haulm lone), was below the standard range of 0.8±1.2 ml suggested by Foote (1980). This low semen volume was probably due to the lower level of protein in the diet resulting in retarded growth and maturity of reproductive organs. This situation, however, is not to be rated adverse since the values were still with in the normal range (0.3±1.6 ml) suggested by Moss et al. (1979). But adverse effects could be noted when protein de®ciency becomes severe enough to cause a live weight loss of about 32% (Parker and Thwaites, 1972).
Values recorded in the supplemented groups were slightly larger compared to those (0.7±0.8 ml) reported by Chiboka (1980) for West African dwarf rams electro-ejaculated weekly but smaller compared to that (1.25 ml) reported by Ihukwumere and Okere (1990) for Nigerian Yankassa rams. These variations could possibly be attributed to either breed, age (Moss et al., 1979) or other environmental factors.
Absence of signi®cant response to increasing levels of leucaena (Fig. 1B) most likely indicates that inclusion up to 300 g per head per day of leucaena in the diet would not have adverse effect on the accessory sex glands of rams.
Fig. 1. Changes in mean values of (A) scrotal circumference; (B) seminal volume (volume); (C) motility percentage; and (D) motility score of spermatozoa of rams offered chickpea haulm basal diet supplemented with 100 (100 g l), 200 (200 g l) and 300 g per head per day (300 g l) of leucaena leaf hay and 300 g per head per day of concentrate.
4.2.2. Spermatozoal motility
Acquisition of progressive motility is considered to be one of the strong evidences for sperm maturation and a useful determinant of conception. The range in the proportions of actively motile spermatozoa recorded in this study was wide (10±76%). Supplementation generally improved the nutritional condition of the animals consequently improving maturation and motility of spermatozoa. This is in agreement with the results of Chiboka (1980), who reported motility of 40% in West African dwarf rams grazing on dry roughage which later increased to 59% with improved grazing conditions. Oldham et al. (1978) also noted signi®cant improvements in spermatogenesis of rams with protein supplementation. In this study, except for rams maintained on chickpea haulm alone, the values were within the standard range (60±80%) set for mature rams (Foote, 1980).
4.2.3. Concentration and number of sperm cells
Despite the variations between treatments, sperm cell concentrations recorded here appeared to be slightly higher compared to the normal range of 1.6±6109 and 2± 3109mlÿ1suggested by Moss et al. (1979) and Foote (1980), respectively, for mature rams. While comparative data in these aspects are lacking in other sheep breeds of Ethiopia, Chiboka (1980) recorded only 0.9109mlÿ1 in the semen of West African dwarf rams under grazing conditions in Nigeria. However, concentration of sperm cells is a highly variable determinant of semen quality (Garner and Hafez, 1980); and thus such disparities in the values reported are supposed to be normal.
Increasing sperm concentration is generally considered to be bene®cial because it allows insemination of a larger number of females (Foote, 1980). During natural mating higher sperm concentration ensures entry of more spermatozoa into the cervical reservoir and then into the oviduct, consequently increasing the chance of fertilization (Garner and Hafez, 1980).
Both leucaena and concentrate supplementation increased the total number of spermatozoa per ejaculate in a manner similar to the seminal volume and sperm cell concentration.
4.2.4. Motility score
Leucaena supplementation has signi®cantly although not consistently improved the mass activity (motility score) of spermatozoa (Fig. 1D). Spermatozoa from rams kept on the roughage diet showed a very slow and spasmodic motion. This is in line with the work of Salamon (1976) who reported signi®cantly higher scores for spermatozoa of rams receiving high level of supplementary feeding (600 g TDN, 165 g CP) compared to those on a lower level of feeding (310 g TDN, 45 g CP). Similarly, Parker and Thwaites (1972) noted the adverse effect of poor nutrition on mass activity of sperm cells in ram semen. The motility scores for spermatozoa of supplemented sheep (2.8±3.3) were comparable to the values reported by Hamani et al. (1996) for White Fulani and Tuareg rams in Niger.
4.2.5. Morphology of sperm cells
According to Moss et al. (1979), this ®gure appears to be out of the normal range (5± 20%). However, there is no concrete evidence to conclude that the semen of these rams is infertile. Certain types of abnormalities may not be associated with infertility, even when the proportions exceed 20% (Foote, 1980). Out of the total defects observed in spermatozoa of non-supplemented rams, only 10% were major defects while the remaining 24% were minor. The latter defects were mainly due to bent or terminally coiled tails, which usually occur during the passage of spermatozoa through the epididymis (Jainudeen and Hafez, 1980). A higher proportion of these abnormalities might be attributed to disturbances in the function of the epididymis probably due to subnormal levels of testosterone (Hainonen, 1989). Production of this hormone was stated to be seriously suppressed as a result of feeding poor quality diets for a prolonged period (Parker and Thwaites, 1972).
Proximal protoplasmic droplets were the major abnormalities encountered in common to all the treatment groups. The greatest (p<0.05) proportion of proximal droplets was observed in spermatozoa of rams on sole chickpea haulm diet (6.5%) which is fairly high, compared to the normal incidence of 2.5% (Ramma and Rao, 1982). It is, however, far below the proportion (30±60%) that would be recorded when testicular degeneration is expected (Ramma and Rao, 1982). Appearance of a relatively larger quantity of protoplasmic droplets probably indicates emission of immature spermatozoa by this group of rams.
4.3. Scrotal circumference (SC)
The results of this experiment indicated that improving DM intake or feeding high quality supplements could enhance testicular growth in rams as indicated by scrotal circumference. This is in agreement with the ®ndings of Oldham et al. (1978) in rams and FAO (1986) in bulls. The SC of rams on the roughage diet even reduced slightly at the end of the experiment (Fig. 1A) probably due to loss of fat in the scrotal tissue (Coulter and Kozub, 1984).
4.4. Correlations
SC was signi®cantly and positively correlated to the volume of ejaculate (V) and total number of spermatozoa per ejaculate (TSPZ), (p<0.01), the coef®cients ranging fromr0.42 to 0.49. Hahn et al. (1969), also found a strong correlation (r0.81) between SC and sperm output in bulls indicating the positive effect of testicular size on sperm production. The correlation observed between SC andV, however, may not necessarily imply ejaculation of larger volumes of semen from bigger testes since the voluminous portion of semen, the seminal plasma, is produced out of the testicle, in the accessory sex glands (Foote, 1980).
5. Conclusions
The ®ndings of this study indicated that supplementation of up to 300 g per head per day of leucaena resulted in improved feed intake, testicular growth and sperm production,
and semen quality of Ethiopian highland sheep offered chickpea haulm basal diet. At the levels tested, no clinical symptoms of leucaena toxicity were apparent and no ill effects were noted on potential fertility of rams.
Acknowledgements
The authors wish to acknowledge the assistance given by the staff of the Animal Science Department at Debre Zeit Agricultural Research Centre, and Dr. Emiru Zewdie of the National Arti®cial Insemination Centre. The technical support rendered by ILRI, Debre Zeit station is highly appreciated. Our due appreciation also goes to Dr. Tilahun Sahlu of the Langiston University, Institute of Goat Research, USA, for supplying us with some very important equipment.
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