Effect of Tribulus terrestris extract on semen quality and serum total cholesterol content in White Plymouth Rock-mini cocks

Svetlana Grigorova, Borislav Kashamov, Veselina Sredkova, Sabka Surdjiiska

Institute of Animal Science - Kostinbrod, Bulgaria

Andrey Kurtenkov

University of Forestry - Sofia, Bulgaria

Introduction

Tribulus terrestris L. (Zygophyllaceae), also called “Puncture vine”, is a prostrate annual herb native of Mediterranean region, but widely distributed in warm regions of Europe, Asia, America, Africa and Australia (Frohne, 1999). This plant has been used in the folk medicine in many countries including Bulgaria for different purposes, but is popularly claimed to improve the sexual function in humans. It contains biologically active substances as steroids, saponins, flavonoids, alkaloids, unsaturated fatty acids, vitamins, tannins, etc. (Adaikan et al., 2000). The main active components of this plant are saponins of the furostanol type, termed protodioscin (Tomova, 1987; Kostova and Dinchev, 2005). Several different samples of plant material were analyzed and depending on origin and plant part used for extraction significant differences in the composition of the saponins were observed; the analysis of market products showed considerable variations of 0.17 to 6.49 % in the protodioscin content (Ganzera et al., 2001).

The clinical investigations carried out in Bulgaria and in other countries show that Tribulus terrestris extract improves the reproductive functions in humans, rats and mice (Tomova, 1987; Viktorov et al., 1994; Gauthaman et al., 2002, 2003; Teuscher and Lindequist, 1994). Kistanova et al. (2005) observed a positive effect of the extract from Tribulus terrestris on the spermatogenesis of rams. Adaikan et al. (2000) studied the proerectile pharmacological effect of Tribulus terrestris extract on the rabbits corpus cavernosum and established stimulatory effect on the erection.

Tribulus terrestris extract is commonly used in the folk medicine also for control of blood pressure and cholesterol. There are reports showing that this extract decreases blood cholesterol level in humans, rats and mice (Li et al., 2001; Chu et al., 2003). There exist no data concerning the effect of Tribulus terrestris extract in poultry.

The aim of this study was to investigate the effect of Tribulus terrestris extract, added to the water of White Plymouth Rock-mini cocks on semen quality and serum total cholesterol content.

Material and Methods

An experiment was carried out in the Poultry Experimental base of the Institute of Animal Sciences-Kostinbrod with twenty cocks at the age of twenty three weeks from the population White Plymouth Rock-mini. They were divided in two groups - control and experimental, ten birds in each. The experiment lasted twenty weeks – one week preparatory and nineteen weeks experimental period.

The cocks from the two groups were fed ad libitum the same diet containing 17.6 % crude protein and 2720 kcal/kg metabolizable energy (Table1). The extract from Tribulus terrestris (producer Vemo Ltd, Sofia, Bulgaria) contained 55% furostanol saponins of which protodioscin is the dominant saponin. This extract was added to the drinking water of the experimental cocks in a dose of 10 mg/kg body weight daily for a period of eleven weeks at the beginning of the experimental period. The next eight weeks of the experimental period were envisaged to measure the aftereffect of Tribulus terrestris.

Semen was collected once weekly during the first four weeks and once per four weeks during the rest period. The sperm was received by the massage method of Burrows and Quin (1938). The following parameters were evaluated: reaction to massage (positive and negative), ejaculating cocks (%), volume (ml), spermatozoids motility (score) concentration of spermatozoids (mlrd/ml), time of methylen blue decolouration (min).

Serum total cholesterol content was determined at the beginning, in the middle and in the end of the trial in both groups of cocks. The blood was taken from vena cutanea ulnaris. The cholesterol content was determined by the method of Watson (1960).

Statistical estimation was conducted by Student’s t-test.

Results and Discussion

The positive massage response in experimental and control cocks during the first test, carried out at the age of twenty three weeks, was 53.3 % and 73.3 % respectively. The observed differences to the massage-induced ejaculation throughout the experiment could be due to the individual stress susceptibility of cocks to the handling (see Table 2).

The age dynamics of the quantitative and qualitative characteristics of ejaculate in both groups was studied.

The most considerable effect of Tribulus terrestris extract was observed on the volume of ejaculate (Fig. 1). During the treatment of the experimental cocks (24-33 week of age) this parameter increased up to 29 % in relation to the control group. Eight weeks after cessation of the treatment ejaculate volume was maintained higher in experimental cocks as compared to that in control cocks.

Sperm motility and concentration in the experimental group were higher (P > 0.05) throughout the experiment (Fig. 2 and Fig. 3).

The time of methylen blue decolouration is presented in Fig. 4. This parameter shows the spermatozoids respiratory activity and depends on their motility and concentration. The experimental cocks, which had a higher spermatozoids concentration and motility were with a shorter time of methylen blue decolouration.

The obtained results are in agreement with the data of Kistanova et.al. (2005), who observed an improvement of the semen quality in rams, given once daily 1.5 g Tribulus terrestris extract per head for a period of forty days.

Viktorov et al. (1994) established positive effect of Tribulus on the spermatogenesis in mice.

The positive effect of Tribulus terrestris on the reproductive system might be explained with the fact, that protodioscin in the Tribulus increases the level of luteinizing hormone, which in its turn stimulates testosterone secretion (Tomova et al., 1981; Antonio et al., 2000).

The data about serum total cholesterol content in the cocks of both groups are pointed in Table 3. The average value of cholesterol level within the experimental period was 9.24 % lower (P > 0.05) in experimental cocks than in control cocks. A great individual variability of this parameter among the cocks in the both groups was observed.

Chu et al. (2003) observed significant decline of serum total cholesterol level in mice, given Tribulus terrestris supplemented diet.

Similar results have been reported by Li et al. (2001) in mice given Tribulus terrestris decoction supplemented diet.

Conclusion

The addition of extract of Tribulus trrestris to the water of White Plymouth Rock–mini cocks had a positive effect on the quantitative and qualitative characteristics of their sperm. Tribulus terrestris increased ejaculate volume, spermatozoids motility, concentration and shortened the time of methylen blue decolouration. Serum total cholesterol content in the experimental cocks was 9.24 % lower (P > 0.05) in comparison with the control group. It is noteworthy that the aftereffect of Tribulus terrestris extract on the studied parameters was maintained for 8 weeks.

Acknowledgment

We would like to thank Mr Hristo Zlatev (Vemo Ltd, Sofia, Bulgaria) for providing the Tribulus terrestris extract that was used in this study.

SUMMARY

The aim of this study was to investigate how the addition of Tribulus terrestris extract to the drinking water of the White Plymouth Rock-mini cocks affects their semen quality and blood serum total cholesterol content. An experiment was conducted with twenty cocks at the age of twenty three weeks from the population White Plymouth Rock-mini, divided in two groups – control and experimental, ten birds each. The trial lasted twenty weeks - one week preparatory and nineteen weeks experimental period. Tribulus terrestris extract was added to the drinking water of the experimental cocks once daily in a dose of 10 mg/kg body weight for a period of eleven weeks. Semen parameters and serum total cholesterol content were evaluated. The sperm was collected by a massage method once weekly during the first four weeks and once per four weeks during the rest period. The age dynamics of the quantitative and qualitative characteristics of ejaculate in both groups of cocks was studied. The addition of Tribulus terrestris extract to the water of the cocks had a positive effect on the sperm quality: increased ejaculate volume, spermatozoids concentration and motility and shortened time of methylen blue decolouration. The serum total cholesterol content in the experimental cocks was 9.24 % lower (P > 0.05) in comparison with the control group. The aftereffect of Tribulus terrestris on the studied parameters was maintained for eight weeks.

Key words: Tribulus terrestris, cocks, semen quality, serum cholesterol

References

1. Adaikan, G.P., K.Gauthaman, R.N.Prasad, C.S.Ng, 2000, Proerektile Pharmacological Effects Of Tr. Terrestris Extract On The Rabbit Corpus Cavernosum. Annals Academy Of Medicine, 29 (1): 22-26.
2. Antonio, J., J.Uelmen, R.Rodriguez, C.Earnest, 2000, The effects of Tr. Terrestris on body composition and exercise performance in resistance – trained males. Int. J. Sport. Nutr. Exerc. Metab., 10 (2): 208-215.
3. Burrows W., J.Quin, 1938, The Collection Of Spermatozoa From The Domestic Fowl And Turkey, Poultry Science, 16 (1): 19-24.
4. Chu S., W.Qu, X.Pang, B.Sun, X.Huang, 2003, Effect of saponin from Tr. terrestris on hyperlipidemia, Zhong Yao Ca, 26 (26): 341-344.
5. Frohne, D., 1999, Ein neues Dopingmittel, Deutsche Apotheker Zeitung, 49: 4752-4754.
6. Ganzera, M., E.Bedir, I.A.Khan, 2001, Determination of steroidal saponins in Tribulus terrestris by reversed-phase high-performance liquid chromatography and evaporative light scattering detection, Journal of Pharmaceutical Sciences, 90 (11): 1752-1758.
7. Gauthaman, K., A.P.Ganesan, R.N.Prasad, 2003, Sexual effects of Puncture vine (Tr. terrestris) extract (protodioscin) an evaluation using a rat model. Journal of Alternative and Complementary Medicine, 9 (2): 257-265.
8. Gauthaman K., P.G.Adaikan, R.N.Prasad, 2002, Aphrodisiac properties of Tr. Terrestris extract (Protodioscin) in normal and castrerted rats. Life Science, August 9, 71 (12): 1385-1396.
9. Kistanova, E., H.Zlatev, V.Karcheva, A.Kolev, 2005, Effect of plant Tr. terrestris extract on reproductive performances of rams, Biotechnology in Animal husbandry, 21 (1-2): 55-63.
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11. LI, M., W.Qu, S.Chu, H.Wang, C.Tian, M.Tu, 2001, Effect of the decoction of Tr. terrestris on mice gluconeogenesis, Zhong Yao Cai, 24 (8): 586-588.
12. Teuscher, E., U.Lindequist, 1994, Biogene Gifte, 2 Auflage, S.163, Wissenschaftliche Verlagsgesellschaft, Stuttgart.
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14. Tomova, M., R.Gyulemetova, S.Zarkova, S.Peeva, T.Panagarova, M.Simona, 1981, Steroidal saponins from Tribulus terrestris L. with a stimulating action on the sexual functions, First Intern. Conf. Chem. Biotechnolog. Biol. Active Nat. Prod., Proceeding, Varna, September 3: 289-291.
15. Viktorov, I., E.Bozadjieva, M.Protich, 1994, Pharmacological, pharmacokinetic, toxicological and clinical studies on protodioscin, IIMS Therapeutic Focus, 213-223.
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Table 1.

Composition and nutritive value of the feed mixture

Components	Percent (%)
Maize	30.00
Wheat	29.80
Sunflower meal	14.00
Soybean meal	14.50
Sunflower oil	2.00
Limestone	7.55
D-C-P	1.60
Salt	0.20
Premix 3k	0.20
L-lysine HCl	0.05
DL-methionine	0.05
Enzyme	0.05
Total:	100.00
Content:
Crude protein	17.60
Lysine	0.70
Methionine + Cystine	0.70
Calcium	3.13
Phosphorus, available	0.41
Metabolizable energy,kcal/kg	2720

Table.2. Ejaculating cocks (%)

Groups	Weeks
	24	25	26	30	34	38	42
control	80.00	46.67	60.00	73.33	73.33	73.33	60.00
experimental	86.66	53.33	46.67	66.66	80.00	86.60	73.33

Table.3. Serum total cholesterol content in cocks (mg%)

Stage	Control	Experimental
At the start of the trial	117.58 ± 9.44	108.94 ± 3.98
In the middle of the trial	88.00 ± 8.01	77.75 ± 5.12
In the end of the trial	86.47 ± 7.30	78.93 ± 5.57

Fig.1. Volume of ejaculate, ml

Fig.2. Motility of sperms, score

Fig.3. Concentration of spermatozoids, mlrd/ml

Fig.4. Time of methylen blue decolouration, min

Эффект экстракта Tribulus terrestris на качество спермы и содержание общего холестерола в сыворотке крови петухов породы Белый Плимутрок-мини

Светлана Григорова, Борислав Кашамов, Веселина Средкова, Сабка Сурджийска

Институт животноводческих наук - Костинброд, Болгария

Андрей Куртенков

Лесотехнический университет - София, Болгария

Резюме

Исследование проводили с целью установить влияние экстракта Tribulus terrestris на качество спермы и содержание общего холестерола в сыворотке крови петухов. Животные были породы Белый Плимутрок-мини, двадцать трех-недельного возраста, распределены в две групы – опытная и контрольная, каждая по десяти птиц. Экспериментальный период – двадцать недель, из которых одна подготвительная. Экстракт Tribulus terrestris добавляли в питьевую воду, один раз в сутки дозой 10 mg/kg живой массы, в продолжении одинадцати недель. Сперму получали с помощью массажа, первые четыри недели раз в недели, а потом через четырех недель до конца двадцатой недели. Сперму оценяли по следующим основным показателям: объем эякулата, подвижность и концентрация сперматозоидов и время обезцвечивания метиленблау. Добавление экстракта привело к позитивному эффекту по этим показателям – наращивание объема, подвижности и концентрации и укорочивание время обезцвечивания. Содержание общего холестерола в сыворотке крови было, в среднем, на 9.24 % ниже (P > 0.05) у опытной группы, по сравнению с контрольной. Последействие применения экстракта Tribulus terrestris на исследованные параметры простиралось на восемь недель после прекращения добавления препарата в питьевую воду.