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Effect of kisspeptin-10, LH and hCG on serum testosterone concentrations in stallions, donkeys and mules
Theriogenology 102 (2017) 75e79
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Effect of kisspeptin-10, LH and hCG on serum testosterone concentrations in stallions, donkeys and mules Rana Waseem Akhtar, Syed Aftab Hussain Shah, Irfan Zia Qureshi* Laboratory of Animal and Human Physiology, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 April 2017 Received in revised form 20 July 2017 Accepted 21 July 2017 Available online 21 July 2017
This study was conducted to determine the response of serum testosterone (T) in male equines (stallions, donkeys and mules) after administering intravenous doses of kisspeptin-10 (KP-10), human chorionic gonadotropin (hCG) and luteinizing hormone (LH) and saline as a control. The animals were divided into four groups of three each: Group I, 3 ml of 0.95% saline; Group II, 50 mg KP-10; Group III, 2500 IU hCG and group IV, 400 mg LH. The administration of KP-10 and hCG to stallions resulted in a significant increase in serum T concentration at 240 min; whereas it was significantly higher at 30, 60, 120, and 240 min with LH treatment as compared to pre-dose concentrations. Both KP-10 and hCG significantly elevated the T concentrations in donkeys at 120 and 240 min, respectively; whereas it was significantly higher at 60, 120, and 240 min with LH treatment as compared to pre-dose concentration. Both KP-10 and LH elevated T in donkeys at 240 min as compared to the control and hCG concentrations. After 120 and 240 min, T concentrations in mules were higher (p < 0.05) with administration of KP-10, hCG and LH as compared to the control. In conclusion, the administration of KP-10, hCG and LH elevate the serum T concentration in normal male equines. It is suggested that KP-10 may be useful in situations where an increase in T is desired. Further work is required to determine the effect of KP-10 on T in male equids with reproductive abnormalities before it can be used in clinical situations. © 2017 Elsevier Inc. All rights reserved.
Keywords: Equines Kisspeptin-10 LH hCG Testosterone
1. Introduction The gonadotrophin secretion in vertebrates is primarily controlled by gonadotrophin-releasing hormone (GnRH), but two other hypothalamic neuropeptides, the gonadotrophin-inhibitory hormone (GnIH) and kisspeptin (KP) have shown a profound control over reproductive functions [1,2]. Kisspeptin and its receptors are vital for activation of the hypothalamic-pituitary-gonadal (HPG) axis in mammals [1,3,4]. Kisspeptin which is a potent endogenous stimulator of GnRH, and the KP neuronal system, directs both the pulsatile GnRH secretion that drives maturation of the ovarian follicle, spermatogenesis and steroid formation of egg cells, and the GnRH surge that activates ovulation in females [5]. Physiological role of kisspeptide in diestrous horse mare is supported by a threshold-like response and an increase in both LH and FSH, when given exogenously. Further neuroanatomical evidence has been generated from experiments that demonstrated kisspeptin
* Corresponding author. E-mail address: [email protected] (I.Z. Qureshi). http://dx.doi.org/10.1016/j.theriogenology.2017.07.027 0093-691X/© 2017 Elsevier Inc. All rights reserved.
immune immunoreactivity (kiSS-ir) throughout the preoptic area (POA) and hypothalamus of the diestrous horse mare with a close association between kiSS-ir fibers and GnRH positive neurons [6]. It has also been reported that KP stimulates the release of testosterone (T) in rodents [7e9] and primates [10]. However, the studies in male equines with regard to the role of KP are still lacking. The aim of developing the human chorionic gonadotropin (hCG) test is to detect functional testicular tissue. It is also helpful for differentiating fully castrated animals from those with retained testes or testicular remnants [11]. It was however reported in a study that no response to GnRH was observed with respect to plasma T in control stallions, aged stallions, and stallions with lack of libido [12]. The male mule (Equus mulus mulus, 63 chromosomes) is a sterile domestic animal that results from the breeding of a male donkey (Equus asinus, 62 chromosomes) to a female horse (Equus caballus, 64 chromosomes), [13,14]. The mechanisms involved in the determination of testis structure and function in mules most likely originated from donkeys. Moreover, the data for mules suggest that their seminiferous tubules are capable of maintaining entire spermatogenesis [15]. Both the luteinizing hormone (LH) and
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hCG are heterodimeric glycoprotein hormones which belong to cystine-knot growth factor families and retain the properties of cytokines and chemokines [16]. Human chorionic gonadotropin, due to its higher receptor binding affinity and a longer circulatory half-life, is more potent than LH [17]. In the stallion, serum LH levels differ with season, with spring levels being 3e5 times higher than early winter [18,19]. The role of KP along with hCG and LH needs to be investigated for the advancement of new strategies for the effective management of fertilization potential in livestock [20]. Therefore, this study was designed to determine the response of serum T in three intact male equines (stallions, donkeys and mules) following intra-venous administration of KP-10, hCG and LH at different intervals of time. The present study, for the first time, aimed to determine the serum T response to KP-10 in stallion, donkey and mule; T response to hCG treatment in the mule and T response to LH treatment in the donkey and mule. We hypothesized that administration of intravenous KP-10, hCG and LH would result in an increase in serum T concentrations in intact male equines. 2. Materials and methods The present study was designed and conducted in the laboratory of Animal and Human Physiology, Department of Animal Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Stallions were housed at a local veterinary farm in Chakwal city. Adult mules were housed at Military Farms, Remount Depot Sargodha. The donkeys were housed at Quaid-i-Azam University, Islamabad. The animals had good body condition score (5e6), having flat back with no crease or ridge. The ribs were not visually distinguishable. Fat around tailhead was spongy. The withers appeared rounded over spinous processes. Shoulders and neck blended smoothly into body [21]. All the animals had palpably normal and descended testes. The testicular morphometric evaluation was performed by caliper measuring length, height, and width, three times a day. Length, width, height, cross-sectional area and circumference at the widest point of the testis were measured by ultrasonography [22]. The volume of the ellipsoid (4/3pabc, a ¼ length/2, b ¼ height/2, c ¼ width/2) was used for predicting the testicular volume. The average length, width, height and testicular volumes of stallions' testes were 14 cm, 8.0 cm, 7.0 cm, and 500 cm3, respectively. The average length, width, height and testicular volumes of donkeys' testes were 10 cm, 6.3 cm, 6.0 cm, and 450 cm3, respectively. The average length, width, height and testicular volumes of mules' testes were 5 cm, 4.3 cm, 4.5 cm, and 50 cm3, respectively. Animal handling and experimental work were done according to the guidelines of the “Bioethics Committee of Faculty of Biological Sciences, Quaid-i-Azam University on Animal Handling and Use for Scientific Research.” 2.1. Experimental design and dose preparation A total of 36 animals were used in the experiment, i.e. 12 animals of each species. The weight range of stallions, donkeys and mules was 300e350, 200e250 and 350e400 kg, while ages were 6e8, 8e10, and 5e6 years, respectively. The experiments were performed at the onset of mating season. Animals of each of the three species were divided into four groups. These included Group I treated with 3 ml of 0.95% normal saline, Group II treated with 50 mg KP-10, Group III treated with 400 mg LH and Group IV treated with 2500 IU hCG. 2.1.1. Dose preparation 2.1.1.1. Kisspeptin-10 preparation. Kisspeptin-10 Human, amino acids 45e54, [Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2]
was obtained from Calbiochem (USA) as 1 mg lyophilized powder. Stock solution was prepared as per instructions of the manufacturer. Kisspeptin-10 dose concentrations were prepared in 0.9% normal saline; 20 equal doses were prepared, while the concentration of each dose was 50 mg. The 50 mg dose was selected following the Wahab et al. [23] who used this dose on adult intact male rhesus monkeys. 2.1.1.2. Luteinizing hormone preparation. Luteinizing hormone dose was prepared according to Schauer et al. [24] with one vial of lyophilized powder containing 75 IU LH (Human, CAS Number 39341-83-8, Sigma-Aldrich, Co. LLC, USA). It was dissolved completely in 1 ml ampoule of sterile and pyrogen free 0.85% injectable sodium chloride. 2.1.1.3. Human chorionic gonadotropin preparation. Human chorionic gonadotropin dose was prepared according to Magee et al. [6]. One vial of lyophilized powder containing 2500 IU of human hCG (CAS RN 9002-61-3, Sigma-Aldrich, Co. LLC) was dissolved completely in 1 ml ampoule of sterile and pyrogen free 0.85% inject able sodium chloride. 2.1.2. Drug administration and blood sampling Pre-dose blood samples were collected from each animal 1 h before the administration of KP-10, hCG, and LH. Doses of KP-10, hCG, and LH and normal saline were administered intravenously into the jugular vein. Animals received each treatment dose only once (n ¼ 3 per species, per treatment). After administering the dose, blood samples (5 ml each) were collected in serum collecting tubes at time intervals of 30, 60, 120, and 240 min, respectively from each animal within a group. Blood was left to clot for 30e40 min at room temperature. Blood samples were centrifuged at 1500 g (22331 Hamburg Centrifuge, Germany) for 15 min at 4 C to obtain the sera. Serum samples were stored at 20 C until experimental analysis. 2.2. Determination of serum testosterone and assay procedure Standard enzyme linked immunosorbent assay was carried out using the commercially available kits (Amgenix, Inc. USA) to determine serum T concentrations at different time intervals for each treatment group in three equids. The testosterone kit contained 96 goat anti-rabbit IgG-coated microtiter wells. The sensitivity of the T assay was 0.05 ng/ml. Interintra-assay coefficient of variations were 12% and 5.5%, respectively. Testosterone reference standards were used at 0, 0.1, 0.5, 2, 6, and 18 ng/ml concentrations. Preferred numbers of labeled wells were placed in the holder. Ten microliters each of standards, samples, and controls were put into suitable wells. Testosterone-horseradish peroxidase (HRP) conjugate (100 ml) was added to each well. Rabbit anti-T mixture (50 ml) was also added to each well. All reagents were mixed thoroughly for 30 s. Wells were incubated at 37 C for 90 min. The microwells were rinsed with deionized water and flicked five times. Tetramethylbenzidine (TMB) reagent (100 ml) was added to each well and was smoothly mixed for 10 s. The wells were then incubated at room temperature (18e25 C) for 20 min. One hundred microliters of stop solution (1N HCl) was used to stop the reaction in all wells. The solution was then mixed gently for 30 s till blue color changed to yellow. The absorbance was read with a 96-well micro plate reader (AMP Diagnostics Platos R 496, Austria) within 10 min. Testosterone concentrations were determined from standard curve (supplementary data) by interpolation using a semi logarithmic curve fit with absorbance on Y axis and T concentration on X axis (ng/ml).
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2.3. Statistical analysis As no interaction between animal species and treatments was observed using general linear model (GLM), comparison of T concentrations among different treatment groups was analyzed by one-way analysis of variance (ANOVA), followed by post-hoc Tukey's test for multiple comparisons within groups. The width values of testes were analyzed with the nonparametric KruskalWallis test because they were not normally distributed. Lengths and heights of testes were analyzed using Student's t-test. The differences were considered significant at p < 0.05 and results are presented as mean ± SEM. The Statistical Package for Social Sciences (SPSS Inc., version 17.0 Chicago. Illinois. USA) was used for the analysis of the data. 3. Results 3.1. T concentrations in stallions The data on the effect of KP-10, hCG, and LH on serum T concentrations in stallions are presented in Table 1. Both KP-10 and hCG administration resulted in a significant increase (p < 0.05) in serum T concentrations at 240 min as compared to pre-dose concentrations. After LH administration, T concentrations were greatly elevated (p < 0.05) at 30, 60, 120, and 240 min time intervals, respectively as compared to the pre-dose concentrations. 3.2. T concentrations in donkeys The data on the effect of KP-10, hCG, and LH on serum T concentrations in donkeys are presented in Table 2. Upon KP-10 treatment, serum T concentrations were significantly increased (p < 0.05) at 120 and 240 min as compared to the pre-dose concentrations. Administration of hCG led to a significant elevation in T concentrations at 120 and 240 min (p < 0.05) as compared to pre Table 1 Effect of Kisspeptin-10 (50 mg KP-10), human chorionic gonadotrophin (2500 IU hCG), and luteinizing hormone (400 mg LH) on serum testosterone concentrations (ng/ml) in stallions as compared to control (3 ml of 0.95% normal saline); (n ¼ 12; 3 stallions per treatment group; intravenous injection). Time Intervals Pre-dose 30 min 60 min 120 min 240 min
Control 1.74 1.82 1.81 2.08 1.85
± ± ± ± ±
0.30 0.33 0.33 0.46 0.38
KP-10 1.50 1.70 1.88 1.91 2.04
± ± ± ± ±
hCG b
0.13 0.13ab 0.12ab 0.11ab 0.04a
1.52 1.98 2.04 2.15 2.33
LH ± ± ± ± ±
b
0.20 0.11ab 0.09ab 0.10ab 0.17a
1.47 1.78 1.91 1.99 2.10
± ± ± ± ±
0.05b 0.04a 0.06a 0.03a 0.07a
Values are shown as mean ± S.E.M. Values without a common superscript in a column show significant (p < 0.05) difference among the treatments as compared to pre-dose at a particular time interval.
Table 2 Effect of Kisspeptin-10 (50 mg KP-10), human chorionic gonadotrophin (2500 IU hCG), and luteinizing hormone (400 mg LH) on serum testosterone concentrations (ng/ml) in donkeys as compared to control (3 ml of 0.95% normal saline); (n ¼ 12; 3 donkeys per treatment group; intravenous injection). Time Intervals
Control
Pre-dose 30 min 60 min 120 min 240 min
1.45 1.54 1.68 1.75 1.72
± ± ± ± ±
0.11 0.13 0.13 0.12 0.10B
KP-10 1.51 1.58 1.63 1.97 2.10
± ± ± ± ±
hCG 0.04b 0.08b 0.06b 0.07a 0.06a,A
1.40 1.44 1.66 1.87 1.98
LH ± ± ± ± ±
0.03b 0.05ab 0.12ab 0.06a 0.05a,B
1.46 1.58 1.72 1.92 2.08
± ± ± ± ±
0.04b 0.08b 0.09a 0.07a 0.06 a,
A
Values are shown as mean ± S.E.M. Values without a common superscript in a row (capital letters) and column (small letters) show a significant (p < 0.05) difference among the treatments at a particular time interval.
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Table 3 Effect of Kisspeptin-10 (50 mg KP-10), human chorionic gonadotrophin (2500 IU hCG), and luteinizing hormone (400 mg LH) on serum testosterone concentrations (ng/ml) in mules as compared to control (3 ml of 0.95% normal saline); (n ¼ 12; 3 mules per treatment group; intravenous injection). Time Intervals Pre-dose 30 min 60 min 120 min 240 min
Control 2.12 2.29 2.32 2.36 2.30
± ± ± ± ±
0.05 0.16 0.15B 0.11B 0.08B
KP-10 2.05 2.36 2.63 2.84 3.04
± ± ± ± ±
hCG b
0.05 0.06a 0.03a, B 0.08a,A 0.10a, A
2.07 2.57 2.78 2.87 2.98
LH ± ± ± ± ±
b
0.04 0.06a 0.07a, A 0.04a,A 0.03a, A
2.05 2.35 2.63 2.79 3.11
± ± ± ± ±
0.01b 0.04a 0.02a, B 0.02a,A 0.07a, A
Values are shown as mean ± S.E.M. Values without a common superscript in a row (capital letters) and column (small letters) show a significant (p < 0.05) difference among the treatments at a particular time interval.
dose concentrations. Administration of LH resulted in a continuous increase (p < 0.05) in serum concentrations of T at 60, 120, and 240 min, respectively as compared to the pre-dose concentrations. The administration of KP-10 and LH elevated concentrations of T (p < 0.05) at 240 min as compared to the control and hCG concentrations in donkeys. 3.3. T concentrations in mules The data on the effect of KP-10, hCG, and LH on serum T concentrations in mules are presented in Table 3. After administering KP-10, hCG and LH, a significant elevation (p < 0.05) in serum T concentrations was observed at all time intervals as compared to pre-dose concentrations. After 60 min, T concentrations were higher (p < 0.05) with administration of hCG as compared to control, KP-10 and LH in mules. At 120 and 240 min, serum T concentrations were higher (p < 0.05) with administration of KP-10, hCG and LH as compared to the control. 4. Discussion The present study was designed to determine the response of serum T in three intact male equines (stallions, donkeys and mules) following intravenous administration of KP-10, hCG and LH at different intervals of time. The present study is the first that determined and compared serum T response after KP-10 administration in stallion, donkey and mule; T response to hCG treatment in the mule and T response to LH treatment in the donkey and mule. Kisspeptin-10 peptide has a complete inherent biological activity [25,26]. The present study demonstrated that administration of KP-10 significantly increases serum T concentrations at 240 min in stallions, 120 and 240 min in donkeys and at all time intervals in mules as compared to pre-dose concentrations. Similar to the results of our study, it has also been reported that an increase in systemic T concentrations occurs following administration of KP-10 in monkeys [27] and male Shiba goats [28]. It was found that acute intravenous administration of KP-10 had stimulatory effects on gonadotropin secretion in men over a dose range of 0.01e1.00 mg/ kg [29,30]. In another study in rats, it was reported that KP-10 significantly increased total plasma T concentrations in the acute phase while chronic administration was associated with a decrease in its concentrations [31]. It has been reported that both Kiss1 and Kiss1r are localized in germ cells and testes of adult rhesus monkey. Kisspeptin 1 expression was evident in round spermatocytes and spermatids, while a co-localization of Kiss1r with inhibin was confirmed in the Sertoli cells. However, Kiss1 and Kiss1r expression were notably absent in Leydig cells. Kisspeptin 1 and Kiss1r expression in testes suggests possible direct involvement in the regulatory network involved in spermatogenesis [32]. Moreover,
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expression and release of KP from mouse testicular Leydig cells has also been demonstrated, and Leydig cell KP expression has been shown to be modulated developmentally by LH [33,34]. This highlights that KP-10 may be administered to stallions for elevation of T concentration as peripheral circulation reflects the endocrine function of the testes [12]. The hCG response test is most reliable for detecting functional testicular tissue. It is useful for assessing testicular function in breeding stallions. It also assists in recognizing fully castrated animals from those with retained testes or testicular remnants [11]. We found that administration of 2500 IU hCG resulted in a significant increase in serum T concentration at 240 min in stallions, 120 and 240 min in donkeys, and at all time intervals in mules as compared to pre-dose concentration. In another study, T concentrations after intravenous injection of 3000 IU hCG at 240 min were 4.5 ± 1.0 ng/ml, 1.6 ± 0.3 ng/ml, and 3.0 ± 1.5 ng/ml in the control, aged and lack of libido stallions, respectively [12]. In contrast to our study, it was reported that the intravenous injection of 12000 IU hCG into horses possessing testicular tissue stimulated a rise in T concentrations which could be detected within 25e35 min of the injection [35]. It has been reported that stallions, cryptorchids and horses with only abdominal testes have lower T concentration in winter as compared to summer [35e37]. It was reported that the hCG test has limited application in horses in the winter [35]. After administering hCG, an elevation in plasma T has also been reported in stallions [38e40]. It was observed that the administration of hCG elevated the plasma T concentrations by stimulating Leydig cells [41]. The role of LH and hCG in the regulation of normal reproductive functions in male and female animals is exclusively established [12,42]. We observed that after LH administration, T concentrations were significantly elevated at all time intervals in stallions and mules while T was elevated at 60, 120, and 240 min in donkeys, respectively as compared to pre-dose concentrations. This demonstrates that LH induced T response earlier in stallions and mules than donkeys. It is very well established that mules are infertile due to chromosomal abnormalities as a result of their parentage preventing normal chromosomal components in gamete production [14]. The nucleolar diameter of Sertoli cells which is reduced in mules as compared to donkeys, is most likely related to the functional position of these cells owing to the occurrence of a smaller amount of germ cells [15]. Moreover, these animals have normal HPG axes and undergo normal puberty. It has been reported that in different model animals, HPG axis is stimulated by central and peripheral treatment of KP [43]. The focus of our study was to observe any variations in serum T concentrations after administering the three hormones in mules, while the fertility test was beyond the scope of current study. Future studies should focus on administering the GnRHR antagonist (antide) prior to KP-10 treatment as an additional group. This would help to understand the independent role of KP on the testes in intact male equids. Kisspeptin-10 is clearly a potent GnRH secretagogue. It is suggested that KP analogues with more powerful GnRH/LH secretion-inducing activity may also be used for the management of fertility in domestic animals [6,20,44,45]. Equally important is to recognize which hormone would be the best choice to test for a T response in equines in a breeding management situation. Overall, it appears that hCG (which is a very potent LH) was not as effective in inducing a response in donkeys as were KP-10 and LH. Another interesting result was that all the three hormones increased T concentrations earlier in the mules than in horses or donkeys. In addition, LH induced a T response earlier in horses than KP-10 or hCG.
4.1. Conclusion In conclusion, administration of KP-10 and hCG and LH elevate serum T concentration in normal male equines. It is suggested that KP-10 may be useful in situations where an increase in T is desired. Further work is required to determine the effect of KP-10 on T in male equids with reproductive abnormalities before it can be used in clinical situations. Authors' contribution First author performed the experimental work, analyzed the data and prepared the first draft. Second author assisted in improving the manuscript through editing, data analysis, intellectual input and bibliography. Third and corresponding author designed the present research work, assisted in analyzing the data and edited the manuscript. Conflict of interest The authors have no conflict of interest with regard to the present study. Acknowledgment The authors thank QAU, Islamabad, Pakistan for providing funds and research facilities to conduct the present study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.theriogenology.2017.07.027. References [1] Tsutsui K, Bentley GE, Kriegsfeld LJ, Osugi T, Seon JY, Vaudry H. Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction. J Neuroendocrinol 2010;22:716e27. [2] Fuqua JS. Treatment and outcomes of precocious puberty: an update. J Clin Endocrinol Metabol 2013;98:2198e207. [3] Saito H, Sawada T, Yaegashi T, Goto Y, Jin J, Sawai K, et al. Kisspeptin-10 stimulates the release of luteinizing hormone and testosterone in pre- and postpubertal male goats. Anim Sci J 2012;83:487e92. [4] Irfan S, Ehmcke J, Wahab F, Shahab M, Schlatt S. Intratesticular action of kisspeptin in rhesus monkey (Macaca mulatta). Andrologia 2014;46:610e7. [5] Tsutsumi R, Webster NJ. GnRH pulsatility, the pituitary response and reproductive dysfunction. Endocr J 2009;56:729e37. [6] Magee C, Foradori CD, Bruemmer JE, Arreguin-Arevalo JA, McCue PM, Handa RJ, et al. Biological and anatomical evidence for kisspeptin regulation of the hypothalamic-pituitary-gonadal axis of estrous horse mares. Endocrinol 2009;150:2813e21. [7] Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, et al. Central and peripheral administration of kisspeptin-10 stimulates the hypothalamicepituitaryegonadal axis. J Neuroendocrinol 2004;16:850e8. [8] Patterson M, Murphy KG, Thompson EL, Patel S, Ghatei MA, Bloom SR. Administration of kisspeptin-54 into discrete regions of the hypothalamus potently increases plasma luteinizing hormone and testosterone in male adult rats. J Neuroendocrinol 2006;18:349e54. [9] Mikkelsen JD, Bentsen AH, Ansel L, Simonneaux V, Juul A. Comparison of the effects of peripherally administered kisspeptins. Regul Pept 2009;152:95e100. [10] Ramaswamy S, Seminara SB, Pohl CR, DiPietrom MJ, Crowleym Jr WF, Plant TM. Effect of continuous intravenous administration of human metastin 45e54 on the neuroendocrine activity of the hypothalamic-pituitarytesticular axis in the adult male rhesus monkey (Macaca mulatta). Endocrinol 2007;148:3364e70. [11] Cox JE, Williams JH. Some aspects of the reproductive endocrinology of the stallion and cryptorchid. J Reprod Fert Suppl 1975:23. [12] Parlevliet JM, Bevers MM, van de Broek J, Colenbrander B. Reproduction: effect of GNRH and HCG administration on plasma LH and testosterone concentrations in normal stallions, aged stallions and stallions with lack of libido. Vet Quart 2001;2001(23):84e7. [13] Benirschke K, Brownhill E, Beath MM. Somatic chromosomes of the horse, the donkey and their hybrids, the mule and the hinny. J Reprod Fertil 1962;4: 319e26.
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hypogonadism in obesity and type 2 diabetes. Neuroendocrinol 2010;91: 302e7. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, et al. Kisspeptin-54 stimulates the hypothalamicepituitaryegonadal axis in human males. J Clin Endocrinol Metabol 2005;90:6609e15. € g ul C, To €re F, Kandirali IE, et al. The effect of Aytürk N, Firat T, Kükner A, Ozo kisspeptin on spermatogenesis and apoptosis in rats. Turk J Med Sci 2017;47: 334e42. Tariq AR, Shahab M, Clarke IJ, Pereira A, Smith JT, Khan Saeed-ul-Hasan, et al. Kiss1 and Kiss1 receptor expression in the rhesus monkey testis: a possible local regulator of testicular function. Cent Eur J Biol 2013;8:968e74. Salehi S, Adeshina I, Chen H, Zirkin BR, Hussain MA, Wondisford F, et al. Developmental and endocrine regulation of kisspeptin expression in mouse Leydig cells. Endocrinol 2015;156:1514e22. Wang J-Y, Hsu M-C, Tseng T-H, Wu L-S, Yang K-T, Chiu C-H. Kisspeptin expression in mouse Leydig cells correlates with age. J Chin Med Assoc 2015;78:249e57. Cox JE, Williams JH, Rowe PH, Smith JA. Testosterone in normal, cryptorchid and castrated male horses. Equine Vet J 1973;5:85e90. Berndtson WE, Pickett BW, Nett TM. Reproductive physiology of the stallion. IV. Seasonal changes in the testosterone concentration of peripheral plasma. J Reprod Fert 1974;39:115e8. Ganjanm VK. Androgens and oestrogens in the normal and cryptorchid stallion. J Reprod Fertil Suppl 1975;23:75e9. Setchell BP, Cox JE. Secretion of free and conjugated steroids by the horse testis into lymph and venous blood. J Reprod Fertil Suppl 1982;32:123e7. Zwain I, Gaillard JL, Dintinger T, Silberzahn P. Down-regulation of testicular aromatization in the horse. Biol Reprod 1989:503e10. Roser JF. Endocrine profiles in fertile, subfertile and infertile stallions: testicular response to human chorionic gonadotropin in infertile stallions. Biol Reprod Mono 1995:661e9. Pickett BW, Amann RP, McKinnon AO, Squires EL, Voss JL. Management of the stallion for maximum reproductive efficiency, II. Colorado State Univ. Animal Reprod 1989;5:73e81. Rahman NA, Rao CV. Recent progress in luteinizing hormone/human chorionic gonadotrophin hormone research. Mol Hum Reprod 2009;15: 703e11. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, et al. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinol 2004;145:4073e7. Curtis AE, Cooke JH, Baxter JE, Parkinson JRC, Bataveljic A, Ghatei MA, et al. A kisspeptin-10 analog with greater in vivo bioactivity than kisspeptin-10. Am J Physiol Endocrinol Metabol 2010;298:E296e303. Matsui H, Tanaka A, Yokoyama K, Takatsu Y, Ishikawa K, Asami T, et al. Chronic administration of the metastin/kisspeptin analog KISS1-305 or the investigational agent TAK-448 suppresses hypothalamic pituitary gonadal function and depletes plasma testosterone in adult male rats. Endocrinol 2012;153:5297e308.
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Effect of kisspeptin-10, LH and hCG on serum testosterone concentrations in stallions, donkeys and mules Rana Waseem Akhtar, Syed Aftab Hussain Shah, Irfan Zia Qureshi* Laboratory of Animal and Human Physiology, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 April 2017 Received in revised form 20 July 2017 Accepted 21 July 2017 Available online 21 July 2017
This study was conducted to determine the response of serum testosterone (T) in male equines (stallions, donkeys and mules) after administering intravenous doses of kisspeptin-10 (KP-10), human chorionic gonadotropin (hCG) and luteinizing hormone (LH) and saline as a control. The animals were divided into four groups of three each: Group I, 3 ml of 0.95% saline; Group II, 50 mg KP-10; Group III, 2500 IU hCG and group IV, 400 mg LH. The administration of KP-10 and hCG to stallions resulted in a significant increase in serum T concentration at 240 min; whereas it was significantly higher at 30, 60, 120, and 240 min with LH treatment as compared to pre-dose concentrations. Both KP-10 and hCG significantly elevated the T concentrations in donkeys at 120 and 240 min, respectively; whereas it was significantly higher at 60, 120, and 240 min with LH treatment as compared to pre-dose concentration. Both KP-10 and LH elevated T in donkeys at 240 min as compared to the control and hCG concentrations. After 120 and 240 min, T concentrations in mules were higher (p < 0.05) with administration of KP-10, hCG and LH as compared to the control. In conclusion, the administration of KP-10, hCG and LH elevate the serum T concentration in normal male equines. It is suggested that KP-10 may be useful in situations where an increase in T is desired. Further work is required to determine the effect of KP-10 on T in male equids with reproductive abnormalities before it can be used in clinical situations. © 2017 Elsevier Inc. All rights reserved.
Keywords: Equines Kisspeptin-10 LH hCG Testosterone
1. Introduction The gonadotrophin secretion in vertebrates is primarily controlled by gonadotrophin-releasing hormone (GnRH), but two other hypothalamic neuropeptides, the gonadotrophin-inhibitory hormone (GnIH) and kisspeptin (KP) have shown a profound control over reproductive functions [1,2]. Kisspeptin and its receptors are vital for activation of the hypothalamic-pituitary-gonadal (HPG) axis in mammals [1,3,4]. Kisspeptin which is a potent endogenous stimulator of GnRH, and the KP neuronal system, directs both the pulsatile GnRH secretion that drives maturation of the ovarian follicle, spermatogenesis and steroid formation of egg cells, and the GnRH surge that activates ovulation in females [5]. Physiological role of kisspeptide in diestrous horse mare is supported by a threshold-like response and an increase in both LH and FSH, when given exogenously. Further neuroanatomical evidence has been generated from experiments that demonstrated kisspeptin
* Corresponding author. E-mail address: [email protected] (I.Z. Qureshi). http://dx.doi.org/10.1016/j.theriogenology.2017.07.027 0093-691X/© 2017 Elsevier Inc. All rights reserved.
immune immunoreactivity (kiSS-ir) throughout the preoptic area (POA) and hypothalamus of the diestrous horse mare with a close association between kiSS-ir fibers and GnRH positive neurons [6]. It has also been reported that KP stimulates the release of testosterone (T) in rodents [7e9] and primates [10]. However, the studies in male equines with regard to the role of KP are still lacking. The aim of developing the human chorionic gonadotropin (hCG) test is to detect functional testicular tissue. It is also helpful for differentiating fully castrated animals from those with retained testes or testicular remnants [11]. It was however reported in a study that no response to GnRH was observed with respect to plasma T in control stallions, aged stallions, and stallions with lack of libido [12]. The male mule (Equus mulus mulus, 63 chromosomes) is a sterile domestic animal that results from the breeding of a male donkey (Equus asinus, 62 chromosomes) to a female horse (Equus caballus, 64 chromosomes), [13,14]. The mechanisms involved in the determination of testis structure and function in mules most likely originated from donkeys. Moreover, the data for mules suggest that their seminiferous tubules are capable of maintaining entire spermatogenesis [15]. Both the luteinizing hormone (LH) and
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hCG are heterodimeric glycoprotein hormones which belong to cystine-knot growth factor families and retain the properties of cytokines and chemokines [16]. Human chorionic gonadotropin, due to its higher receptor binding affinity and a longer circulatory half-life, is more potent than LH [17]. In the stallion, serum LH levels differ with season, with spring levels being 3e5 times higher than early winter [18,19]. The role of KP along with hCG and LH needs to be investigated for the advancement of new strategies for the effective management of fertilization potential in livestock [20]. Therefore, this study was designed to determine the response of serum T in three intact male equines (stallions, donkeys and mules) following intra-venous administration of KP-10, hCG and LH at different intervals of time. The present study, for the first time, aimed to determine the serum T response to KP-10 in stallion, donkey and mule; T response to hCG treatment in the mule and T response to LH treatment in the donkey and mule. We hypothesized that administration of intravenous KP-10, hCG and LH would result in an increase in serum T concentrations in intact male equines. 2. Materials and methods The present study was designed and conducted in the laboratory of Animal and Human Physiology, Department of Animal Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Stallions were housed at a local veterinary farm in Chakwal city. Adult mules were housed at Military Farms, Remount Depot Sargodha. The donkeys were housed at Quaid-i-Azam University, Islamabad. The animals had good body condition score (5e6), having flat back with no crease or ridge. The ribs were not visually distinguishable. Fat around tailhead was spongy. The withers appeared rounded over spinous processes. Shoulders and neck blended smoothly into body [21]. All the animals had palpably normal and descended testes. The testicular morphometric evaluation was performed by caliper measuring length, height, and width, three times a day. Length, width, height, cross-sectional area and circumference at the widest point of the testis were measured by ultrasonography [22]. The volume of the ellipsoid (4/3pabc, a ¼ length/2, b ¼ height/2, c ¼ width/2) was used for predicting the testicular volume. The average length, width, height and testicular volumes of stallions' testes were 14 cm, 8.0 cm, 7.0 cm, and 500 cm3, respectively. The average length, width, height and testicular volumes of donkeys' testes were 10 cm, 6.3 cm, 6.0 cm, and 450 cm3, respectively. The average length, width, height and testicular volumes of mules' testes were 5 cm, 4.3 cm, 4.5 cm, and 50 cm3, respectively. Animal handling and experimental work were done according to the guidelines of the “Bioethics Committee of Faculty of Biological Sciences, Quaid-i-Azam University on Animal Handling and Use for Scientific Research.” 2.1. Experimental design and dose preparation A total of 36 animals were used in the experiment, i.e. 12 animals of each species. The weight range of stallions, donkeys and mules was 300e350, 200e250 and 350e400 kg, while ages were 6e8, 8e10, and 5e6 years, respectively. The experiments were performed at the onset of mating season. Animals of each of the three species were divided into four groups. These included Group I treated with 3 ml of 0.95% normal saline, Group II treated with 50 mg KP-10, Group III treated with 400 mg LH and Group IV treated with 2500 IU hCG. 2.1.1. Dose preparation 2.1.1.1. Kisspeptin-10 preparation. Kisspeptin-10 Human, amino acids 45e54, [Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2]
was obtained from Calbiochem (USA) as 1 mg lyophilized powder. Stock solution was prepared as per instructions of the manufacturer. Kisspeptin-10 dose concentrations were prepared in 0.9% normal saline; 20 equal doses were prepared, while the concentration of each dose was 50 mg. The 50 mg dose was selected following the Wahab et al. [23] who used this dose on adult intact male rhesus monkeys. 2.1.1.2. Luteinizing hormone preparation. Luteinizing hormone dose was prepared according to Schauer et al. [24] with one vial of lyophilized powder containing 75 IU LH (Human, CAS Number 39341-83-8, Sigma-Aldrich, Co. LLC, USA). It was dissolved completely in 1 ml ampoule of sterile and pyrogen free 0.85% injectable sodium chloride. 2.1.1.3. Human chorionic gonadotropin preparation. Human chorionic gonadotropin dose was prepared according to Magee et al. [6]. One vial of lyophilized powder containing 2500 IU of human hCG (CAS RN 9002-61-3, Sigma-Aldrich, Co. LLC) was dissolved completely in 1 ml ampoule of sterile and pyrogen free 0.85% inject able sodium chloride. 2.1.2. Drug administration and blood sampling Pre-dose blood samples were collected from each animal 1 h before the administration of KP-10, hCG, and LH. Doses of KP-10, hCG, and LH and normal saline were administered intravenously into the jugular vein. Animals received each treatment dose only once (n ¼ 3 per species, per treatment). After administering the dose, blood samples (5 ml each) were collected in serum collecting tubes at time intervals of 30, 60, 120, and 240 min, respectively from each animal within a group. Blood was left to clot for 30e40 min at room temperature. Blood samples were centrifuged at 1500 g (22331 Hamburg Centrifuge, Germany) for 15 min at 4 C to obtain the sera. Serum samples were stored at 20 C until experimental analysis. 2.2. Determination of serum testosterone and assay procedure Standard enzyme linked immunosorbent assay was carried out using the commercially available kits (Amgenix, Inc. USA) to determine serum T concentrations at different time intervals for each treatment group in three equids. The testosterone kit contained 96 goat anti-rabbit IgG-coated microtiter wells. The sensitivity of the T assay was 0.05 ng/ml. Interintra-assay coefficient of variations were 12% and 5.5%, respectively. Testosterone reference standards were used at 0, 0.1, 0.5, 2, 6, and 18 ng/ml concentrations. Preferred numbers of labeled wells were placed in the holder. Ten microliters each of standards, samples, and controls were put into suitable wells. Testosterone-horseradish peroxidase (HRP) conjugate (100 ml) was added to each well. Rabbit anti-T mixture (50 ml) was also added to each well. All reagents were mixed thoroughly for 30 s. Wells were incubated at 37 C for 90 min. The microwells were rinsed with deionized water and flicked five times. Tetramethylbenzidine (TMB) reagent (100 ml) was added to each well and was smoothly mixed for 10 s. The wells were then incubated at room temperature (18e25 C) for 20 min. One hundred microliters of stop solution (1N HCl) was used to stop the reaction in all wells. The solution was then mixed gently for 30 s till blue color changed to yellow. The absorbance was read with a 96-well micro plate reader (AMP Diagnostics Platos R 496, Austria) within 10 min. Testosterone concentrations were determined from standard curve (supplementary data) by interpolation using a semi logarithmic curve fit with absorbance on Y axis and T concentration on X axis (ng/ml).
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2.3. Statistical analysis As no interaction between animal species and treatments was observed using general linear model (GLM), comparison of T concentrations among different treatment groups was analyzed by one-way analysis of variance (ANOVA), followed by post-hoc Tukey's test for multiple comparisons within groups. The width values of testes were analyzed with the nonparametric KruskalWallis test because they were not normally distributed. Lengths and heights of testes were analyzed using Student's t-test. The differences were considered significant at p < 0.05 and results are presented as mean ± SEM. The Statistical Package for Social Sciences (SPSS Inc., version 17.0 Chicago. Illinois. USA) was used for the analysis of the data. 3. Results 3.1. T concentrations in stallions The data on the effect of KP-10, hCG, and LH on serum T concentrations in stallions are presented in Table 1. Both KP-10 and hCG administration resulted in a significant increase (p < 0.05) in serum T concentrations at 240 min as compared to pre-dose concentrations. After LH administration, T concentrations were greatly elevated (p < 0.05) at 30, 60, 120, and 240 min time intervals, respectively as compared to the pre-dose concentrations. 3.2. T concentrations in donkeys The data on the effect of KP-10, hCG, and LH on serum T concentrations in donkeys are presented in Table 2. Upon KP-10 treatment, serum T concentrations were significantly increased (p < 0.05) at 120 and 240 min as compared to the pre-dose concentrations. Administration of hCG led to a significant elevation in T concentrations at 120 and 240 min (p < 0.05) as compared to pre Table 1 Effect of Kisspeptin-10 (50 mg KP-10), human chorionic gonadotrophin (2500 IU hCG), and luteinizing hormone (400 mg LH) on serum testosterone concentrations (ng/ml) in stallions as compared to control (3 ml of 0.95% normal saline); (n ¼ 12; 3 stallions per treatment group; intravenous injection). Time Intervals Pre-dose 30 min 60 min 120 min 240 min
Control 1.74 1.82 1.81 2.08 1.85
± ± ± ± ±
0.30 0.33 0.33 0.46 0.38
KP-10 1.50 1.70 1.88 1.91 2.04
± ± ± ± ±
hCG b
0.13 0.13ab 0.12ab 0.11ab 0.04a
1.52 1.98 2.04 2.15 2.33
LH ± ± ± ± ±
b
0.20 0.11ab 0.09ab 0.10ab 0.17a
1.47 1.78 1.91 1.99 2.10
± ± ± ± ±
0.05b 0.04a 0.06a 0.03a 0.07a
Values are shown as mean ± S.E.M. Values without a common superscript in a column show significant (p < 0.05) difference among the treatments as compared to pre-dose at a particular time interval.
Table 2 Effect of Kisspeptin-10 (50 mg KP-10), human chorionic gonadotrophin (2500 IU hCG), and luteinizing hormone (400 mg LH) on serum testosterone concentrations (ng/ml) in donkeys as compared to control (3 ml of 0.95% normal saline); (n ¼ 12; 3 donkeys per treatment group; intravenous injection). Time Intervals
Control
Pre-dose 30 min 60 min 120 min 240 min
1.45 1.54 1.68 1.75 1.72
± ± ± ± ±
0.11 0.13 0.13 0.12 0.10B
KP-10 1.51 1.58 1.63 1.97 2.10
± ± ± ± ±
hCG 0.04b 0.08b 0.06b 0.07a 0.06a,A
1.40 1.44 1.66 1.87 1.98
LH ± ± ± ± ±
0.03b 0.05ab 0.12ab 0.06a 0.05a,B
1.46 1.58 1.72 1.92 2.08
± ± ± ± ±
0.04b 0.08b 0.09a 0.07a 0.06 a,
A
Values are shown as mean ± S.E.M. Values without a common superscript in a row (capital letters) and column (small letters) show a significant (p < 0.05) difference among the treatments at a particular time interval.
77
Table 3 Effect of Kisspeptin-10 (50 mg KP-10), human chorionic gonadotrophin (2500 IU hCG), and luteinizing hormone (400 mg LH) on serum testosterone concentrations (ng/ml) in mules as compared to control (3 ml of 0.95% normal saline); (n ¼ 12; 3 mules per treatment group; intravenous injection). Time Intervals Pre-dose 30 min 60 min 120 min 240 min
Control 2.12 2.29 2.32 2.36 2.30
± ± ± ± ±
0.05 0.16 0.15B 0.11B 0.08B
KP-10 2.05 2.36 2.63 2.84 3.04
± ± ± ± ±
hCG b
0.05 0.06a 0.03a, B 0.08a,A 0.10a, A
2.07 2.57 2.78 2.87 2.98
LH ± ± ± ± ±
b
0.04 0.06a 0.07a, A 0.04a,A 0.03a, A
2.05 2.35 2.63 2.79 3.11
± ± ± ± ±
0.01b 0.04a 0.02a, B 0.02a,A 0.07a, A
Values are shown as mean ± S.E.M. Values without a common superscript in a row (capital letters) and column (small letters) show a significant (p < 0.05) difference among the treatments at a particular time interval.
dose concentrations. Administration of LH resulted in a continuous increase (p < 0.05) in serum concentrations of T at 60, 120, and 240 min, respectively as compared to the pre-dose concentrations. The administration of KP-10 and LH elevated concentrations of T (p < 0.05) at 240 min as compared to the control and hCG concentrations in donkeys. 3.3. T concentrations in mules The data on the effect of KP-10, hCG, and LH on serum T concentrations in mules are presented in Table 3. After administering KP-10, hCG and LH, a significant elevation (p < 0.05) in serum T concentrations was observed at all time intervals as compared to pre-dose concentrations. After 60 min, T concentrations were higher (p < 0.05) with administration of hCG as compared to control, KP-10 and LH in mules. At 120 and 240 min, serum T concentrations were higher (p < 0.05) with administration of KP-10, hCG and LH as compared to the control. 4. Discussion The present study was designed to determine the response of serum T in three intact male equines (stallions, donkeys and mules) following intravenous administration of KP-10, hCG and LH at different intervals of time. The present study is the first that determined and compared serum T response after KP-10 administration in stallion, donkey and mule; T response to hCG treatment in the mule and T response to LH treatment in the donkey and mule. Kisspeptin-10 peptide has a complete inherent biological activity [25,26]. The present study demonstrated that administration of KP-10 significantly increases serum T concentrations at 240 min in stallions, 120 and 240 min in donkeys and at all time intervals in mules as compared to pre-dose concentrations. Similar to the results of our study, it has also been reported that an increase in systemic T concentrations occurs following administration of KP-10 in monkeys [27] and male Shiba goats [28]. It was found that acute intravenous administration of KP-10 had stimulatory effects on gonadotropin secretion in men over a dose range of 0.01e1.00 mg/ kg [29,30]. In another study in rats, it was reported that KP-10 significantly increased total plasma T concentrations in the acute phase while chronic administration was associated with a decrease in its concentrations [31]. It has been reported that both Kiss1 and Kiss1r are localized in germ cells and testes of adult rhesus monkey. Kisspeptin 1 expression was evident in round spermatocytes and spermatids, while a co-localization of Kiss1r with inhibin was confirmed in the Sertoli cells. However, Kiss1 and Kiss1r expression were notably absent in Leydig cells. Kisspeptin 1 and Kiss1r expression in testes suggests possible direct involvement in the regulatory network involved in spermatogenesis [32]. Moreover,
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expression and release of KP from mouse testicular Leydig cells has also been demonstrated, and Leydig cell KP expression has been shown to be modulated developmentally by LH [33,34]. This highlights that KP-10 may be administered to stallions for elevation of T concentration as peripheral circulation reflects the endocrine function of the testes [12]. The hCG response test is most reliable for detecting functional testicular tissue. It is useful for assessing testicular function in breeding stallions. It also assists in recognizing fully castrated animals from those with retained testes or testicular remnants [11]. We found that administration of 2500 IU hCG resulted in a significant increase in serum T concentration at 240 min in stallions, 120 and 240 min in donkeys, and at all time intervals in mules as compared to pre-dose concentration. In another study, T concentrations after intravenous injection of 3000 IU hCG at 240 min were 4.5 ± 1.0 ng/ml, 1.6 ± 0.3 ng/ml, and 3.0 ± 1.5 ng/ml in the control, aged and lack of libido stallions, respectively [12]. In contrast to our study, it was reported that the intravenous injection of 12000 IU hCG into horses possessing testicular tissue stimulated a rise in T concentrations which could be detected within 25e35 min of the injection [35]. It has been reported that stallions, cryptorchids and horses with only abdominal testes have lower T concentration in winter as compared to summer [35e37]. It was reported that the hCG test has limited application in horses in the winter [35]. After administering hCG, an elevation in plasma T has also been reported in stallions [38e40]. It was observed that the administration of hCG elevated the plasma T concentrations by stimulating Leydig cells [41]. The role of LH and hCG in the regulation of normal reproductive functions in male and female animals is exclusively established [12,42]. We observed that after LH administration, T concentrations were significantly elevated at all time intervals in stallions and mules while T was elevated at 60, 120, and 240 min in donkeys, respectively as compared to pre-dose concentrations. This demonstrates that LH induced T response earlier in stallions and mules than donkeys. It is very well established that mules are infertile due to chromosomal abnormalities as a result of their parentage preventing normal chromosomal components in gamete production [14]. The nucleolar diameter of Sertoli cells which is reduced in mules as compared to donkeys, is most likely related to the functional position of these cells owing to the occurrence of a smaller amount of germ cells [15]. Moreover, these animals have normal HPG axes and undergo normal puberty. It has been reported that in different model animals, HPG axis is stimulated by central and peripheral treatment of KP [43]. The focus of our study was to observe any variations in serum T concentrations after administering the three hormones in mules, while the fertility test was beyond the scope of current study. Future studies should focus on administering the GnRHR antagonist (antide) prior to KP-10 treatment as an additional group. This would help to understand the independent role of KP on the testes in intact male equids. Kisspeptin-10 is clearly a potent GnRH secretagogue. It is suggested that KP analogues with more powerful GnRH/LH secretion-inducing activity may also be used for the management of fertility in domestic animals [6,20,44,45]. Equally important is to recognize which hormone would be the best choice to test for a T response in equines in a breeding management situation. Overall, it appears that hCG (which is a very potent LH) was not as effective in inducing a response in donkeys as were KP-10 and LH. Another interesting result was that all the three hormones increased T concentrations earlier in the mules than in horses or donkeys. In addition, LH induced a T response earlier in horses than KP-10 or hCG.
4.1. Conclusion In conclusion, administration of KP-10 and hCG and LH elevate serum T concentration in normal male equines. It is suggested that KP-10 may be useful in situations where an increase in T is desired. Further work is required to determine the effect of KP-10 on T in male equids with reproductive abnormalities before it can be used in clinical situations. Authors' contribution First author performed the experimental work, analyzed the data and prepared the first draft. Second author assisted in improving the manuscript through editing, data analysis, intellectual input and bibliography. Third and corresponding author designed the present research work, assisted in analyzing the data and edited the manuscript. Conflict of interest The authors have no conflict of interest with regard to the present study. Acknowledgment The authors thank QAU, Islamabad, Pakistan for providing funds and research facilities to conduct the present study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.theriogenology.2017.07.027. References [1] Tsutsui K, Bentley GE, Kriegsfeld LJ, Osugi T, Seon JY, Vaudry H. Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction. J Neuroendocrinol 2010;22:716e27. [2] Fuqua JS. Treatment and outcomes of precocious puberty: an update. J Clin Endocrinol Metabol 2013;98:2198e207. [3] Saito H, Sawada T, Yaegashi T, Goto Y, Jin J, Sawai K, et al. Kisspeptin-10 stimulates the release of luteinizing hormone and testosterone in pre- and postpubertal male goats. Anim Sci J 2012;83:487e92. [4] Irfan S, Ehmcke J, Wahab F, Shahab M, Schlatt S. Intratesticular action of kisspeptin in rhesus monkey (Macaca mulatta). Andrologia 2014;46:610e7. [5] Tsutsumi R, Webster NJ. GnRH pulsatility, the pituitary response and reproductive dysfunction. Endocr J 2009;56:729e37. [6] Magee C, Foradori CD, Bruemmer JE, Arreguin-Arevalo JA, McCue PM, Handa RJ, et al. Biological and anatomical evidence for kisspeptin regulation of the hypothalamic-pituitary-gonadal axis of estrous horse mares. Endocrinol 2009;150:2813e21. [7] Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, et al. Central and peripheral administration of kisspeptin-10 stimulates the hypothalamicepituitaryegonadal axis. J Neuroendocrinol 2004;16:850e8. [8] Patterson M, Murphy KG, Thompson EL, Patel S, Ghatei MA, Bloom SR. Administration of kisspeptin-54 into discrete regions of the hypothalamus potently increases plasma luteinizing hormone and testosterone in male adult rats. J Neuroendocrinol 2006;18:349e54. [9] Mikkelsen JD, Bentsen AH, Ansel L, Simonneaux V, Juul A. Comparison of the effects of peripherally administered kisspeptins. Regul Pept 2009;152:95e100. [10] Ramaswamy S, Seminara SB, Pohl CR, DiPietrom MJ, Crowleym Jr WF, Plant TM. Effect of continuous intravenous administration of human metastin 45e54 on the neuroendocrine activity of the hypothalamic-pituitarytesticular axis in the adult male rhesus monkey (Macaca mulatta). Endocrinol 2007;148:3364e70. [11] Cox JE, Williams JH. Some aspects of the reproductive endocrinology of the stallion and cryptorchid. J Reprod Fert Suppl 1975:23. [12] Parlevliet JM, Bevers MM, van de Broek J, Colenbrander B. Reproduction: effect of GNRH and HCG administration on plasma LH and testosterone concentrations in normal stallions, aged stallions and stallions with lack of libido. Vet Quart 2001;2001(23):84e7. [13] Benirschke K, Brownhill E, Beath MM. Somatic chromosomes of the horse, the donkey and their hybrids, the mule and the hinny. J Reprod Fertil 1962;4: 319e26.
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