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The effects of palm kernel cake based diet on spermatogenesis in Malin × Santa-Ines rams
Animal Reproduction Science 115 (2009) 182–188
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The effects of palm kernel cake based diet on spermatogenesis in Malin × Santa-Ines rams H. Yaakub a,d,∗, M. Masnindah a, G. Shanthi b, S. Sukardi c, A.R. Alimon a,d a b c d
Department of Animal Science, Faculty of Agriculture, Selangor, Malaysia Faculty of Veterinary Medicine, Selangor, Malaysia Faculty of Medicine and Health Sciences, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 7 September 2007 Received in revised form 28 November 2008 Accepted 3 December 2008 Available online 7 December 2008 Keywords: Rams Palm kernel cake Copper toxicity Spermatogenesis Ammonium molybdate
a b s t r a c t Testes from nine male Malin × Santa-Ines rams with an average body weight of 43.1 ± 3.53 kg, were used to study the effects of palm kernel cake (PKC) based diet on spermatogenic cells and to assess copper (Cu) levels in liver, testis and plasma in sheep. Animals were divided into three groups and randomly assigned three dietary treatments using restricted randomization of body weight in completely randomized design. The dietary treatments were 60% palm kernel cake plus 40% oil palm frond (PKC), 60% palm kernel cake plus 40% oil palm frond supplemented with 23 mg/kg dry matter of molybdenum as ammonium molybdate [(NH4 )6 Mo7 O24 ·4H2 O] and 600 mg/kg dry matter of sulphate as sodium sulphate [Na2 SO4 ] (PKC-MS) and 60% concentrate of corn-soybean mix + 40% oil palm frond (Control), the concentrate was mixed in a ratio of 79% corn, 20% soybean meal and 1% standard mineral mix. The results obtained showed that the number of spermatogonia, spermatocytes, spermatids and Leydig cells were not significantly different among the three treatment groups. However, spermatozoa, Sertoli cells and degenerated cells showed significant changes, which, may be probably due to the Cu content in PKC. Liver and testis Cu levels in the rams under PKC diet was found to be significantly higher (P < 0.05) than rams in Control and PKC-MS diets. Plasma Cu levels showed a significant increase (P < 0.05) at the end of the experiment as compared to at the beginning of the experiment for PKC and Control. In conclusion, spermatogenesis is normal in
∗ Corresponding author at: Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia. Tel.: +60 3 8946 6894; fax: +60 3 8943 2954. E-mail address: [email protected] (H. Yaakub). 0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.12.006
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rams fed the diet without PKC and PKC supplemented with Mo and S. However spermatogenesis was altered in the PKC based diet probably due to the toxic effects of Cu and the significant changes in organs and plasma. Thus, Mo and S play a major role in reducing the accumulation of Cu in organs. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Spermatogenesis is the entire process of spermatozoon formation. It does not occur simultaneously in all seminiferous tubules but rather in wave-like sequences of maturation, referred to as cycles of the seminiferous epithelium (Gartner et al., 1998). Several studies indicated the effects of imbalance levels of trace minerals to spermatogenesis, i.e., Skandhan (1992) and Vrzgulova et al. (1993) noted that the presence of abnormal levels of copper (Cu) might affect spermatogenesis through production, maturation, motility and fertilizing capacity of the spermatozoa. Palm kernel cake (PKC) is an important by-product of the oil palm industry and is obtained after the extraction of oil from the kernel of the oil palm fruit. It has a high nutritive value and is widely used as a feed ingredient in ruminants. However, PKC has high Cu content and that makes it less suitable for sheep. Abdul Rahman et al. (1989) and Wan Mohamed et al. (1989) reported that excess usage in sheep could cause chronic toxicity due to the accumulation of copper in the liver. The addition of molybdenum (Mo) and sulphur (S) in the PKC diet has been reported to overcome the Cu toxicity problems in rams (Suttle et al., 1984; Abdul Rahman et al., 1989; Goonerate et al., 1989; Kincaid, 1999; Ivan et al., 1999 and Pott et al., 1999). Although the addition of Mo and S in the PKC diets might solved the toxicity problem, but none of the study was conducted to monitor the changes in the histology of the testis. Hence, the objective of this study is to observe the effects of PKC on histological structure of testis in sheep. 2. Materials and methods 2.1. Animals Nine male Malin × Santa-Ines rams were fed with formulated palm kernel cake (n = 3), palm kernel cake plus molybdenum as ammonium molybdate (NH4 )6 Mo7 O24 ·4H2 O) and sulphur as sodium sulphate (Na2 SO4 ) (PKC-MS; n = 3) and Control diet (C; n = 3) for 6 months using a complete randomized design. The nutritional analyses of diets (Table 1) are similar for all types with the exception of the treatments added. The mineral contents (Cu, Zinc, Mo and S) in the diets are presented in Table 2. 2.2. Blood sampling Blood samples for plasma Cu concentrations were collected at the beginning of the experiment and at slaughter into a tube containing anticoagulant (Vacutainer®, Becton Dickinson Limited, England). Table 1 Diet treatments containing palm kernel cake in palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets fed to Malin × Santa-Ines rams. Contents
PKC
PKC-MS
Control
Palm kernel cake Oil palm frond Corn-soybean mix Molybdenum supplement (ppm) Sodium sulphate supplement (ppm)
60% 40% – – –
60% 40% – 23 600
– 40% 60% – –
Formulated as NRC (1985): isonitrogenous (13.5% CP) and isocaloric (2.3 ME Mcal/kg).
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Table 2 Mineral content (mean ± se) in palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets fed to Malin × Santa-Ines rams. Contents
PKC
PKC-MS
Control
Copper (ppm) Zinc (ppm) Molybdenum supplement (ppm) Sulfur supplement (ppm)
11.4 ± 0.3 44.5 0.8 – –
11.4 ± 0.3 44.5 0.8 23 600
7.3 ± 0.9 80.5 1.2 – –
Blood samples were immediately centrifuged at 3000 rpm for 15 min and plasma was separated and stored at −21 ◦ C until analyzed. 2.3. Testis and liver sampling Both testes and liver were removed after slaughter and fresh samples taken for Cu concentrations and stored at −21 ◦ C until analyzed. Testis samples were taken from the mid-region near the rete testis while liver samples from the right lobes were randomised between animals in the same group. Samples were fixed in Bouin’s solution for histology. 2.4. Copper determination All samples were digested with nitric acid (wet oxidation procedure) to digest organic materials and diluted with deionized distilled water as reported by Hair-Bejo and Alimon (1995). Briefly, samples were digested in a pyrex glass tube (150 mm × 18 mm) with 70% aristar grade nitric acid (BDH Chemicals Ltd) and 60% spectrosol grade perchloric acid (BDH Chemicals Ltd.) in 2 to 1 (v/v), respectively. Approximately 50 l of 1 mg/ml of spectrosol grade cupric nitrate were added, covered with glass marbles and left overnight. Heating at 140 ◦ C was carried out on a heating block until all samples were completely digested. Further dilution in distilled water was then followed by measurement of copper contents using inductively coupled plasma method at 324.7 nm in the Atomic Absorption Spectrophotometer. 2.5. Histological preparation and evaluation Fixed testis samples underwent a series of dehydration and clearing processes using the histokinette (Shandon®, Citadel 20000) and embedded in paraffin to form paraffin blocks and serially sectioned using the microtome (Leica® RM 2145) to the thickness of 5 m. Ten slides from each testis samples were randomly selected and stained using hematoxylin and eosin staining procedure. Three areas of seminiferous tubule from each slide were randomly subjected to a quantitative histological evaluation. An ocular graticule fixed to the eyepiece of a normal light microscope (Olympus, UK) was used for detection of spermatogonia, spermatocyte, spermatid, spermatozoa, Sertoli cell, Leydig cells and degenerating cells. Based on the areas of the tubule, spermatogonia should be round and numerous and situated at the periphery of the basal compartment. Spermatocytes have darker nuclei and situated further from the periphery of the tubule. Large nuclei of the Sertoli cells are also found between rows of spematogonia. Early and late spermatids possess a tail and are situated further towards the adluminal compartment while spermatozoa will have their heads embedded in the cytoplasm of Sertoli cells and their tails directed towards the lumen. Degenerating cells would have undergone necrosis. Approximately, 100 cells were counted per field under ×40 magnification. The data will be presented as the mean percentage from three areas per slide of different spermatogenic cells stages in the seminiferous tubule. 2.6. Data analysis One-way analysis of variance (ANOVA) was used to analyze the data for liver and testis weight, Cu concentration and the histological parameters followed by Tukey’s multiple range tests to assess
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Table 3 Blood plasma copper concentrations (mean ± se) at the beginning of experiment and at slaughter in Malin × Santa-Ines rams fed on palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets. Copper concentrations (ppm)
PKC (n = 3)
PKC-MS (n = 3)
Control (n = 3)
Beginning of the experiment At slaughter
0.26 ± 0.08ax 0.63 ± 0.18bx
0.31 ± 0.04ax 0.30 ± 0.03ay
0.24 ± 0.03ax 0.41 ± 0.04by
a,b x,y
Means with different superscripts within the same column are significantly different (P < 0.05). Means with different superscripts within the same row are significantly different (P < 0.05).
Table 4 Liver and testis copper concentrations (mean ± se) at slaughter in Malin × Santa-Ines rams fed on palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets. Organs Liver (ppm) Testis (ppm) a,b
PKC (n = 3) 1089.1 ± 188.9a 10.6 ± 0.7a
PKC-MS (n = 3)
Control (n = 3)
539.4 ± 43.4b 6.6 ± 0.5b
548.9 ± 25.6b 8.2 ± 0.6ab
Means with different superscripts within the same row are significantly different (P < 0.05).
differences among diets (SPSS, 2002). Two-way analysis of variance (ANOVA) was used to analyze copper concentrations in blood. An alpha probability at the 0.05 levels was considered to be statistically significant. 3. Results There were no significant changes in blood plasma Cu concentrations at the beginning of the experiment and slaughter in rams fed PKC-MS (Table 3). There was a significant increase of blood plasma Cu concentration two-fold in PKC diet rams and one-fold in Control diet rams at the beginning of the experiment and at slaughter. At slaughter, the Cu liver concentration was significantly high in rams fed on PKC as compared to PKC-MS and Control diets (Table 4). The Cu concentration was found to be significantly high in rams fed PKC as compared to PKC-MS but however record a non-significant difference with Control diets. Histological observation of testis from rams fed on PKC revealed inhibition of spermatogenesis, where no spermatozoon was found in the seminiferous tubules (Fig. 1A). Both testes from rams fed on PKC-MS and Control showed normal spermatogenesis with spermatogenic cells and interstitial tissues consisting of Leydig cells (Fig. 1B) and with spermatogenic cells at different stages of development (Fig. 1C), respectively. There were also no significant difference in the mean number of spermatogonia, spermatocyte, spermatid and Leydig cells in seminiferous tubules of testes from rams fed on PKC, PKC-MS and Control diets (Table 5). The mean number of spermatozoa and Sertoli cells were significantly lower in seminiferous tubules of testes recovered from rams fed on PKC as compared to PKC-MS and Control.
Table 5 The number (mean ± se) and percentage (%) of different spermatogenic cell stages, Sertoli cells, Leydig cells, degenerating cells in seminiferous tubules in Malin × Santa-Ines testis fed on palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets. Type of cells
PKC
Spermatogonia Spermatocyte Spermatid Spermatozoa Sertoli cells Leydig cells Degenerative cells
22.9 15.7 23.9 2.7 2.1 15.3 16.6
a,b
PKC-MS ± ± ± ± ± ± ±
1.34 (22.8) 1.38 (15.7) 2.08 (24.8) 0.75b (2.7) 0.16b (2.1) 1.82 (15.3) 1.26a (16.6)
13.1 23.7 39.3 9.7 5.6 3.28 5.4
± ± ± ± ± ± ±
0.51 (13.0) 0.84 (23.7) 1.50 (39.3) 1.20 a (9.7) 0.24a (5.6) 0.48 (3.3) 0.57b (5.4)
Means with different superscripts between the column are significantly different (P < 0.05).
Control 13.6 ± 1.75 (13.6) 22.1 ± 0.79 (22.1) 50.0 ± 10.64 (48.7) 6.2 ± 0.71 a (6.1) 4.4 ± 0.23a (4.4) 1.7 ± 0.23 (1.7) 3.5 ± 0.41b (3.4)
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Fig. 1. (A) Cross-section of a testis collected from rams fed on PKC diet. There are no spermatozoa present in the lumen (arrow) of the somniferous tubules but Sertoli cells (SC) are normal (×40 magnifications). (B) Cross-section of testis collected from rams fed on PKC-MS diet. A normal spermatogenesis with spermatogenic cells (Spermatogonia: S1; Spermatocytes: S2; Spermatids: S3 and Spermatozoa: S4) in seminiferous tubules and Leydig cells (LC) outside tubule (×40 magnification). (C) Cross-section of testis collected from rams fed on Control diet. A normal spermatogenesis with spermatogenic cells at different stages of development (Spermatogonia: S1; Spermatocytes: S2; Spermatids: S3 and Spermatozoa: S4) in seminiferous tubules and Leydig cells (LC) outside tubule (×40 magnification).
However, the mean number of degenerated cells was higher in seminiferous tubules of testes recovered from rams fed on PKC than that on PKC-MS or Control diets. 4. Discussion In this study, histological characteristics of testes were evaluated to investigate effects of PKC on spermatogenesis. Additionally, the study also observed the parameters of Cu concentrations in plasma, liver and testis in relation to spermatogenesis. Spermatogenesis is a process that involves the transformation of the undifferentiated immature spermatozoa (Clearmont, 1972). In the present study, spermatogonia and Leydig cells were not affected by any dietary treatments (PKC, PKC-MS and Control). However, spermatocytes and spermatids were found to be the dominant cells affected with no or very few cells in different tubules and in different cases suggesting an arrest in normal development to spermatid stage (spermacytogenesis) or failure of differentiation to spermatozoa (spermiogenesis).
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Spermatozoa, Sertoli and degenerated cells were shown to have significant changes in diets supplemented with PKC. This phenomenon may be due to the increased Cu present during the various stages of sperm development and may be related to Cu levels in the liver, testis and plasma as shown in Table 3 and Table 4. Palm kernel cake diet, which contain 11.4 ppm Cu (Table 2) is sufficient to show an effect on the number of spermatogonia when compared with Control (7.3 ppm). However PKC-MS recorded a higher number of spermatozoa as compared to Control. This suggests that diets supplemented with Mo and S play a major role in reducing the accumulation of Cu in these organs as evidenced by the consistent plasma Cu levels of animals under diet PKC-MS throughout the experimental period. Our results agree with those reported by Hair-Bejo and Alimon (1992) where supplementing 500 ppm Zn or ammonium molybdate relieves Cu toxicity in small ruminants. Sertoli cells also showed significant changes for PKC, PKC-MS and Control diets. A small number of Sertoli cells (2.1 ± 0.16) are found in PKC while a bigger number (5.6 ± 0.24) was found in PKC-MS when compared to Control (4.4 ± 0.23). Degenerated cells were also higher in animals fed PKC (16.6 ± 1.26) as compared to Control where very few tubules were degenerated (1.7 ± 0.23). It is not unusual to find degenerated tubular epithelial cells in normal testis (Ladds, 1993) and the degenerative changes may be due to the toxic effects of Cu on the seminiferous tubules Kimberling (1988). The seminiferous tubule is extremely sensitive to harmful and toxic influences arising in the animal or in its environment as a consequence of which spermatogenesis quickly ceases and the epithelium degenerates (Arthur, 1979) as degenerated cells are the most common lesion associated with acquired infertility and lowered seminal quality (Faulkner and Carroll, 1974). Disruption of the germinal epithelium into the lumen with high degenerative changes to Sertoli cells and spermatogenic cells causing insufficient nutrition and total arrest for further development has also been reported in rams intoxicated with copper from industrial emissions (Vrzgulova et al., 1993). The results obtained for this preliminary experiment using a small number of animals per group is very encouraging and can be useful for the formulation of feed using agricultural by products like palm kernel cake in a larger number of animals in the future. 5. Conclusion Spermatogenesis is normal in rams fed diets without PKC and PKC supplemented with Mo and S, however it was altered probably due to the toxic effects of Cu in the PKC based on the significant changes in organs, seminiferous epithelium and plasma. Thus, diets consisting PKC should be supplemented with Mo and S to reduce the accumulation of Cu in organs especially the testes while simultaneously saving on feed and optimising animal reproductive potentials. Acknowledgements The authors would like to thank the Department of Animal Sciences, Faculty of Agriculture, Universiti Putra Malaysia and MOSTI. References Abdul Rahman, M.Y., Wong, H.K., Zaini, H., Sharif, H., 1989. Preliminary observation on the alleviation of copper in sheep fed with palm kernel meal based diet. In: Proc. of 12th Conf. MSAP, pp. 75–78. Arthur, G.H., 1979. Testicular Degeneration. Veterinary Reproduction and Obstetrics, 4th edition. Bailliere Tindall, London, pp. 570–575. Clearmont, Y., 1972. Kinetics of spermatogenesis in mammals seminiferous epithelium cycle and spermatogonial renewal. Physiol. Rev. 52, 198–265. Faulkner, L.C., Carroll, E.J., 1974. Reproductive failure in males. In: Hafez, E.S.E. (Ed.), Reproduction in Farm Animal. Lea and Febiger, Philadelphia, pp. 373–383. Gartner, L.P., Hiatt, J.L., Strum, J.M., 1998. Cell Biology and Histology, 3rd edition. Lippincott Williams & Wilkins, Maryland, USA, p. 376. Goonerate, S.R., Cuckley, W.T., Christensen, D.A., 1989. Review of copper deficiency and metabolism in ruminants. Can. J. Anim. Sci. 69, 819–845. Hair-Bejo, M., Alimon, A.R., 1992. Hepatic damages and the protective role of zinc and molybdate in Palm Kernel Cake, toxicity in sheep. In: Proc. 15th Conf. MSAP, 26–27 May 1992, Kuala Terengganu, Malaysia, pp. 93–95. Hair-Bejo, M., Alimon, A.R., 1995. The Protective role of zinc in Palm Kernel Cake toxicity in sheep. Mal. J. Nutr. 1, 75–82. Kimberling, C.V., 1988. Jensen and Swift’s Disease of Sheep, 3rd edition. Lea and Febiger, Philadelphia, pp. 372–374. Kincaid, R.L., 1999. Assessment of trace mineral status of ruminants: a review. Proc. Am. Soc. Anim. Sci., 1–10.
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Ladds, P.W., 1993. The male genital system. In: Jubb, K.V.F., Kennedy, P.C., Palmer, N. (Eds.), Pathology of Domestic Animal, 34th edition. Academic Press, San Diego, CA, pp. 471–529. Nutrient Requirements of Sheep, 1985. Sixth Revised Edition. Pott, E.B., Henry, P.R., Rao, P.V., Hinderberger, J., Ammerman, C.B., 1999. Estimated relative bioavailabity of supplemental in organic molybdenum sources and their effect on tissue molybdenum an copper concentration in lambs. Anim. Feeds Tech. 79, 107–117. Skandhan, K.P., 1992. Review on copper in male reproduction and contraception. In: Francaise, R. (Ed.), Gynecol Obstet (Review), vol. 87. pp. 594–598. Suttle, N.F., Abrahams, P., Thornton, I., 1984. The role of a soil X dietary sulphur interaction in the impairment of copper absorption by soil ingestion in sheep. J. Agric. Sci. 103, 81–86. Vrzgulova, M., Bires, J., Vrzgula, L., 1993. The Effect of copper from industrial emissions on the seminiferous epithelium in rams. Reprod. Dom. Anim. 28, 108–118. Wan Mohamed, W.E., Abdul Rahman, A., Mobamad, N., Koh, H.F., 1989. Oil palm by-products in prime lamb production. Proc. of 12th Conf. MSA, P69–P74.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The effects of palm kernel cake based diet on spermatogenesis in Malin × Santa-Ines rams H. Yaakub a,d,∗, M. Masnindah a, G. Shanthi b, S. Sukardi c, A.R. Alimon a,d a b c d
Department of Animal Science, Faculty of Agriculture, Selangor, Malaysia Faculty of Veterinary Medicine, Selangor, Malaysia Faculty of Medicine and Health Sciences, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 7 September 2007 Received in revised form 28 November 2008 Accepted 3 December 2008 Available online 7 December 2008 Keywords: Rams Palm kernel cake Copper toxicity Spermatogenesis Ammonium molybdate
a b s t r a c t Testes from nine male Malin × Santa-Ines rams with an average body weight of 43.1 ± 3.53 kg, were used to study the effects of palm kernel cake (PKC) based diet on spermatogenic cells and to assess copper (Cu) levels in liver, testis and plasma in sheep. Animals were divided into three groups and randomly assigned three dietary treatments using restricted randomization of body weight in completely randomized design. The dietary treatments were 60% palm kernel cake plus 40% oil palm frond (PKC), 60% palm kernel cake plus 40% oil palm frond supplemented with 23 mg/kg dry matter of molybdenum as ammonium molybdate [(NH4 )6 Mo7 O24 ·4H2 O] and 600 mg/kg dry matter of sulphate as sodium sulphate [Na2 SO4 ] (PKC-MS) and 60% concentrate of corn-soybean mix + 40% oil palm frond (Control), the concentrate was mixed in a ratio of 79% corn, 20% soybean meal and 1% standard mineral mix. The results obtained showed that the number of spermatogonia, spermatocytes, spermatids and Leydig cells were not significantly different among the three treatment groups. However, spermatozoa, Sertoli cells and degenerated cells showed significant changes, which, may be probably due to the Cu content in PKC. Liver and testis Cu levels in the rams under PKC diet was found to be significantly higher (P < 0.05) than rams in Control and PKC-MS diets. Plasma Cu levels showed a significant increase (P < 0.05) at the end of the experiment as compared to at the beginning of the experiment for PKC and Control. In conclusion, spermatogenesis is normal in
∗ Corresponding author at: Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia. Tel.: +60 3 8946 6894; fax: +60 3 8943 2954. E-mail address: [email protected] (H. Yaakub). 0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.12.006
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rams fed the diet without PKC and PKC supplemented with Mo and S. However spermatogenesis was altered in the PKC based diet probably due to the toxic effects of Cu and the significant changes in organs and plasma. Thus, Mo and S play a major role in reducing the accumulation of Cu in organs. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Spermatogenesis is the entire process of spermatozoon formation. It does not occur simultaneously in all seminiferous tubules but rather in wave-like sequences of maturation, referred to as cycles of the seminiferous epithelium (Gartner et al., 1998). Several studies indicated the effects of imbalance levels of trace minerals to spermatogenesis, i.e., Skandhan (1992) and Vrzgulova et al. (1993) noted that the presence of abnormal levels of copper (Cu) might affect spermatogenesis through production, maturation, motility and fertilizing capacity of the spermatozoa. Palm kernel cake (PKC) is an important by-product of the oil palm industry and is obtained after the extraction of oil from the kernel of the oil palm fruit. It has a high nutritive value and is widely used as a feed ingredient in ruminants. However, PKC has high Cu content and that makes it less suitable for sheep. Abdul Rahman et al. (1989) and Wan Mohamed et al. (1989) reported that excess usage in sheep could cause chronic toxicity due to the accumulation of copper in the liver. The addition of molybdenum (Mo) and sulphur (S) in the PKC diet has been reported to overcome the Cu toxicity problems in rams (Suttle et al., 1984; Abdul Rahman et al., 1989; Goonerate et al., 1989; Kincaid, 1999; Ivan et al., 1999 and Pott et al., 1999). Although the addition of Mo and S in the PKC diets might solved the toxicity problem, but none of the study was conducted to monitor the changes in the histology of the testis. Hence, the objective of this study is to observe the effects of PKC on histological structure of testis in sheep. 2. Materials and methods 2.1. Animals Nine male Malin × Santa-Ines rams were fed with formulated palm kernel cake (n = 3), palm kernel cake plus molybdenum as ammonium molybdate (NH4 )6 Mo7 O24 ·4H2 O) and sulphur as sodium sulphate (Na2 SO4 ) (PKC-MS; n = 3) and Control diet (C; n = 3) for 6 months using a complete randomized design. The nutritional analyses of diets (Table 1) are similar for all types with the exception of the treatments added. The mineral contents (Cu, Zinc, Mo and S) in the diets are presented in Table 2. 2.2. Blood sampling Blood samples for plasma Cu concentrations were collected at the beginning of the experiment and at slaughter into a tube containing anticoagulant (Vacutainer®, Becton Dickinson Limited, England). Table 1 Diet treatments containing palm kernel cake in palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets fed to Malin × Santa-Ines rams. Contents
PKC
PKC-MS
Control
Palm kernel cake Oil palm frond Corn-soybean mix Molybdenum supplement (ppm) Sodium sulphate supplement (ppm)
60% 40% – – –
60% 40% – 23 600
– 40% 60% – –
Formulated as NRC (1985): isonitrogenous (13.5% CP) and isocaloric (2.3 ME Mcal/kg).
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Table 2 Mineral content (mean ± se) in palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets fed to Malin × Santa-Ines rams. Contents
PKC
PKC-MS
Control
Copper (ppm) Zinc (ppm) Molybdenum supplement (ppm) Sulfur supplement (ppm)
11.4 ± 0.3 44.5 0.8 – –
11.4 ± 0.3 44.5 0.8 23 600
7.3 ± 0.9 80.5 1.2 – –
Blood samples were immediately centrifuged at 3000 rpm for 15 min and plasma was separated and stored at −21 ◦ C until analyzed. 2.3. Testis and liver sampling Both testes and liver were removed after slaughter and fresh samples taken for Cu concentrations and stored at −21 ◦ C until analyzed. Testis samples were taken from the mid-region near the rete testis while liver samples from the right lobes were randomised between animals in the same group. Samples were fixed in Bouin’s solution for histology. 2.4. Copper determination All samples were digested with nitric acid (wet oxidation procedure) to digest organic materials and diluted with deionized distilled water as reported by Hair-Bejo and Alimon (1995). Briefly, samples were digested in a pyrex glass tube (150 mm × 18 mm) with 70% aristar grade nitric acid (BDH Chemicals Ltd) and 60% spectrosol grade perchloric acid (BDH Chemicals Ltd.) in 2 to 1 (v/v), respectively. Approximately 50 l of 1 mg/ml of spectrosol grade cupric nitrate were added, covered with glass marbles and left overnight. Heating at 140 ◦ C was carried out on a heating block until all samples were completely digested. Further dilution in distilled water was then followed by measurement of copper contents using inductively coupled plasma method at 324.7 nm in the Atomic Absorption Spectrophotometer. 2.5. Histological preparation and evaluation Fixed testis samples underwent a series of dehydration and clearing processes using the histokinette (Shandon®, Citadel 20000) and embedded in paraffin to form paraffin blocks and serially sectioned using the microtome (Leica® RM 2145) to the thickness of 5 m. Ten slides from each testis samples were randomly selected and stained using hematoxylin and eosin staining procedure. Three areas of seminiferous tubule from each slide were randomly subjected to a quantitative histological evaluation. An ocular graticule fixed to the eyepiece of a normal light microscope (Olympus, UK) was used for detection of spermatogonia, spermatocyte, spermatid, spermatozoa, Sertoli cell, Leydig cells and degenerating cells. Based on the areas of the tubule, spermatogonia should be round and numerous and situated at the periphery of the basal compartment. Spermatocytes have darker nuclei and situated further from the periphery of the tubule. Large nuclei of the Sertoli cells are also found between rows of spematogonia. Early and late spermatids possess a tail and are situated further towards the adluminal compartment while spermatozoa will have their heads embedded in the cytoplasm of Sertoli cells and their tails directed towards the lumen. Degenerating cells would have undergone necrosis. Approximately, 100 cells were counted per field under ×40 magnification. The data will be presented as the mean percentage from three areas per slide of different spermatogenic cells stages in the seminiferous tubule. 2.6. Data analysis One-way analysis of variance (ANOVA) was used to analyze the data for liver and testis weight, Cu concentration and the histological parameters followed by Tukey’s multiple range tests to assess
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Table 3 Blood plasma copper concentrations (mean ± se) at the beginning of experiment and at slaughter in Malin × Santa-Ines rams fed on palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets. Copper concentrations (ppm)
PKC (n = 3)
PKC-MS (n = 3)
Control (n = 3)
Beginning of the experiment At slaughter
0.26 ± 0.08ax 0.63 ± 0.18bx
0.31 ± 0.04ax 0.30 ± 0.03ay
0.24 ± 0.03ax 0.41 ± 0.04by
a,b x,y
Means with different superscripts within the same column are significantly different (P < 0.05). Means with different superscripts within the same row are significantly different (P < 0.05).
Table 4 Liver and testis copper concentrations (mean ± se) at slaughter in Malin × Santa-Ines rams fed on palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets. Organs Liver (ppm) Testis (ppm) a,b
PKC (n = 3) 1089.1 ± 188.9a 10.6 ± 0.7a
PKC-MS (n = 3)
Control (n = 3)
539.4 ± 43.4b 6.6 ± 0.5b
548.9 ± 25.6b 8.2 ± 0.6ab
Means with different superscripts within the same row are significantly different (P < 0.05).
differences among diets (SPSS, 2002). Two-way analysis of variance (ANOVA) was used to analyze copper concentrations in blood. An alpha probability at the 0.05 levels was considered to be statistically significant. 3. Results There were no significant changes in blood plasma Cu concentrations at the beginning of the experiment and slaughter in rams fed PKC-MS (Table 3). There was a significant increase of blood plasma Cu concentration two-fold in PKC diet rams and one-fold in Control diet rams at the beginning of the experiment and at slaughter. At slaughter, the Cu liver concentration was significantly high in rams fed on PKC as compared to PKC-MS and Control diets (Table 4). The Cu concentration was found to be significantly high in rams fed PKC as compared to PKC-MS but however record a non-significant difference with Control diets. Histological observation of testis from rams fed on PKC revealed inhibition of spermatogenesis, where no spermatozoon was found in the seminiferous tubules (Fig. 1A). Both testes from rams fed on PKC-MS and Control showed normal spermatogenesis with spermatogenic cells and interstitial tissues consisting of Leydig cells (Fig. 1B) and with spermatogenic cells at different stages of development (Fig. 1C), respectively. There were also no significant difference in the mean number of spermatogonia, spermatocyte, spermatid and Leydig cells in seminiferous tubules of testes from rams fed on PKC, PKC-MS and Control diets (Table 5). The mean number of spermatozoa and Sertoli cells were significantly lower in seminiferous tubules of testes recovered from rams fed on PKC as compared to PKC-MS and Control.
Table 5 The number (mean ± se) and percentage (%) of different spermatogenic cell stages, Sertoli cells, Leydig cells, degenerating cells in seminiferous tubules in Malin × Santa-Ines testis fed on palm kernel cake (PKC), palm kernel cake plus molybdenum and sulphur (PKC-MS) and Control diets. Type of cells
PKC
Spermatogonia Spermatocyte Spermatid Spermatozoa Sertoli cells Leydig cells Degenerative cells
22.9 15.7 23.9 2.7 2.1 15.3 16.6
a,b
PKC-MS ± ± ± ± ± ± ±
1.34 (22.8) 1.38 (15.7) 2.08 (24.8) 0.75b (2.7) 0.16b (2.1) 1.82 (15.3) 1.26a (16.6)
13.1 23.7 39.3 9.7 5.6 3.28 5.4
± ± ± ± ± ± ±
0.51 (13.0) 0.84 (23.7) 1.50 (39.3) 1.20 a (9.7) 0.24a (5.6) 0.48 (3.3) 0.57b (5.4)
Means with different superscripts between the column are significantly different (P < 0.05).
Control 13.6 ± 1.75 (13.6) 22.1 ± 0.79 (22.1) 50.0 ± 10.64 (48.7) 6.2 ± 0.71 a (6.1) 4.4 ± 0.23a (4.4) 1.7 ± 0.23 (1.7) 3.5 ± 0.41b (3.4)
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Fig. 1. (A) Cross-section of a testis collected from rams fed on PKC diet. There are no spermatozoa present in the lumen (arrow) of the somniferous tubules but Sertoli cells (SC) are normal (×40 magnifications). (B) Cross-section of testis collected from rams fed on PKC-MS diet. A normal spermatogenesis with spermatogenic cells (Spermatogonia: S1; Spermatocytes: S2; Spermatids: S3 and Spermatozoa: S4) in seminiferous tubules and Leydig cells (LC) outside tubule (×40 magnification). (C) Cross-section of testis collected from rams fed on Control diet. A normal spermatogenesis with spermatogenic cells at different stages of development (Spermatogonia: S1; Spermatocytes: S2; Spermatids: S3 and Spermatozoa: S4) in seminiferous tubules and Leydig cells (LC) outside tubule (×40 magnification).
However, the mean number of degenerated cells was higher in seminiferous tubules of testes recovered from rams fed on PKC than that on PKC-MS or Control diets. 4. Discussion In this study, histological characteristics of testes were evaluated to investigate effects of PKC on spermatogenesis. Additionally, the study also observed the parameters of Cu concentrations in plasma, liver and testis in relation to spermatogenesis. Spermatogenesis is a process that involves the transformation of the undifferentiated immature spermatozoa (Clearmont, 1972). In the present study, spermatogonia and Leydig cells were not affected by any dietary treatments (PKC, PKC-MS and Control). However, spermatocytes and spermatids were found to be the dominant cells affected with no or very few cells in different tubules and in different cases suggesting an arrest in normal development to spermatid stage (spermacytogenesis) or failure of differentiation to spermatozoa (spermiogenesis).
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Spermatozoa, Sertoli and degenerated cells were shown to have significant changes in diets supplemented with PKC. This phenomenon may be due to the increased Cu present during the various stages of sperm development and may be related to Cu levels in the liver, testis and plasma as shown in Table 3 and Table 4. Palm kernel cake diet, which contain 11.4 ppm Cu (Table 2) is sufficient to show an effect on the number of spermatogonia when compared with Control (7.3 ppm). However PKC-MS recorded a higher number of spermatozoa as compared to Control. This suggests that diets supplemented with Mo and S play a major role in reducing the accumulation of Cu in these organs as evidenced by the consistent plasma Cu levels of animals under diet PKC-MS throughout the experimental period. Our results agree with those reported by Hair-Bejo and Alimon (1992) where supplementing 500 ppm Zn or ammonium molybdate relieves Cu toxicity in small ruminants. Sertoli cells also showed significant changes for PKC, PKC-MS and Control diets. A small number of Sertoli cells (2.1 ± 0.16) are found in PKC while a bigger number (5.6 ± 0.24) was found in PKC-MS when compared to Control (4.4 ± 0.23). Degenerated cells were also higher in animals fed PKC (16.6 ± 1.26) as compared to Control where very few tubules were degenerated (1.7 ± 0.23). It is not unusual to find degenerated tubular epithelial cells in normal testis (Ladds, 1993) and the degenerative changes may be due to the toxic effects of Cu on the seminiferous tubules Kimberling (1988). The seminiferous tubule is extremely sensitive to harmful and toxic influences arising in the animal or in its environment as a consequence of which spermatogenesis quickly ceases and the epithelium degenerates (Arthur, 1979) as degenerated cells are the most common lesion associated with acquired infertility and lowered seminal quality (Faulkner and Carroll, 1974). Disruption of the germinal epithelium into the lumen with high degenerative changes to Sertoli cells and spermatogenic cells causing insufficient nutrition and total arrest for further development has also been reported in rams intoxicated with copper from industrial emissions (Vrzgulova et al., 1993). The results obtained for this preliminary experiment using a small number of animals per group is very encouraging and can be useful for the formulation of feed using agricultural by products like palm kernel cake in a larger number of animals in the future. 5. Conclusion Spermatogenesis is normal in rams fed diets without PKC and PKC supplemented with Mo and S, however it was altered probably due to the toxic effects of Cu in the PKC based on the significant changes in organs, seminiferous epithelium and plasma. Thus, diets consisting PKC should be supplemented with Mo and S to reduce the accumulation of Cu in organs especially the testes while simultaneously saving on feed and optimising animal reproductive potentials. Acknowledgements The authors would like to thank the Department of Animal Sciences, Faculty of Agriculture, Universiti Putra Malaysia and MOSTI. References Abdul Rahman, M.Y., Wong, H.K., Zaini, H., Sharif, H., 1989. Preliminary observation on the alleviation of copper in sheep fed with palm kernel meal based diet. In: Proc. of 12th Conf. MSAP, pp. 75–78. Arthur, G.H., 1979. Testicular Degeneration. Veterinary Reproduction and Obstetrics, 4th edition. Bailliere Tindall, London, pp. 570–575. Clearmont, Y., 1972. Kinetics of spermatogenesis in mammals seminiferous epithelium cycle and spermatogonial renewal. Physiol. Rev. 52, 198–265. Faulkner, L.C., Carroll, E.J., 1974. Reproductive failure in males. In: Hafez, E.S.E. (Ed.), Reproduction in Farm Animal. Lea and Febiger, Philadelphia, pp. 373–383. Gartner, L.P., Hiatt, J.L., Strum, J.M., 1998. Cell Biology and Histology, 3rd edition. Lippincott Williams & Wilkins, Maryland, USA, p. 376. Goonerate, S.R., Cuckley, W.T., Christensen, D.A., 1989. Review of copper deficiency and metabolism in ruminants. Can. J. Anim. Sci. 69, 819–845. Hair-Bejo, M., Alimon, A.R., 1992. Hepatic damages and the protective role of zinc and molybdate in Palm Kernel Cake, toxicity in sheep. In: Proc. 15th Conf. MSAP, 26–27 May 1992, Kuala Terengganu, Malaysia, pp. 93–95. Hair-Bejo, M., Alimon, A.R., 1995. The Protective role of zinc in Palm Kernel Cake toxicity in sheep. Mal. J. Nutr. 1, 75–82. Kimberling, C.V., 1988. Jensen and Swift’s Disease of Sheep, 3rd edition. Lea and Febiger, Philadelphia, pp. 372–374. Kincaid, R.L., 1999. Assessment of trace mineral status of ruminants: a review. Proc. Am. Soc. Anim. Sci., 1–10.
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