Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen

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Theriogenology 78 (2012) 907–913 www.theriojournal.com

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen R.T. Padilhaa,*, D.M. Magalhães-Padilhab, M.M. Cavalcantec, A.P. Almeidab, K.T. Haagd, M.O. Gastald, J.F. Nunesc, A.P.R. Rodriguesb, J.R. Figueiredob, M.A.L. Oliveiraa a

Rede Nordeste de Biotecnologia (RENORBIO), Universidade Federal Rural de Pernambuco, Dom Manoel de Medeiros Street, Dois Irmãos, Recife 52171-900, PE, Brazil b Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles (LAMOFOPA), Veterinary Faculty, State University of Ceará, Av. Paranjana, 1700, Campus do Itaperi, Fortaleza 60740 – 000, CE, Brazil c Laboratory of caprine and ovine semen Technology (LTSCO), State University of Ceará, Av. Paranjana, 1700, Campus do Itaperi, Fortaleza 60740 – 000, CE, Brazil d Department of Animal Science, Food and Nutrition, Southern Illinois University Carbondale, 1205 Lincoln Drive, MC 4417, Carbondale, Illinois 62901, USA Received 2 September 2011; received in revised form 2 April 2012; accepted 2 April 2012

Abstract The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P ⬍ 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P ⬍ 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P ⬎ 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P ⬎ 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen. © 2012 Elsevier Inc. All rights reserved. Keywords: Frozen-thawed sperm; IGF-I; Ram semen; Semen cryopreservation; Artificial insemination

1. Introduction * Corresponding author Tel: ⫹55 85 3275 7657; Fax: ⫹55 85 3279 1902. E-mail address: [email protected] (R.T. Padilha). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.04.005

Semen cryopreservation has been widely used to conserve endangered species and/or animals of high genetic value [1], and is well established for some

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domestic animals [2,3] and some nondomestic mammals [4,5]. However, in sheep, fertility after insemination with frozen/thawed semen is relatively low [6]. Poor fertility following AI of frozen/thawed semen in this species could be due to the unsaturated: saturated fatty acids ratio and the proportion of cholesterol in the sperm plasma membrane [7], which could affect the quality of frozen/thawed semen. Semen cryopreservation includes dilution, cryoprotection, cooling and freezing, storage, and thawing, all with the potential to affect sperm structure and function [8]. In general, cooling from body temperature to near freezing causes substantial stress to the sperm plasma membrane, resulting in the rearrangement and destabilization of membrane components and a calcium influx [9 –12]. The extent to which such membrane damage is responsible for the deleterious effects of cryopreservation on sperm motility is unclear [9,13,14]. In an attempt to improve the cryopreservation process, various sperm diluents and substances have been tested [15,16]. IGF-I may improve sperm survivability after a freezethaw cycle by stimulating the synthesis of RNA, protein, and lipids, thereby improving plasma membrane function [15]. It was reported that IGF-I is an import regulator of spermatogenesis [17], acting as a potent mitogenic, metabolic, and differentiating polypeptide. In sexually active stallions, total sperm motility tended to be higher in animals with elevated IGF-I concentrations in their seminal plasma [18]. In addition, pregnancy rates were higher with semen used from horses with high concentrations of IGF-I in their seminal plasma, implicating IGF-I in sperm function [18]. Furthermore, in one study, IGF-I significantly improved both total and progressive motility of bovine sperm in vitro [2]. Moreover, IGF-I has a role in sperm maturation [19] and capacitation [20] in humans, it prevented deterioration of sperm functional parameters and fertility in buffalo [21], and it increased sperm motility and viability in rabbits and bulls [22,23]. Although the importance of IGF-I on some semen quality traits has been reported in some species, the effect of this growth factor as a supplement in semen extender on seminal parameters of ovine frozen/thawed semen and pregnancy rates in ewes after laparoscopic AI has apparently not been reported. Thus, the aim of the present study was to determine if various concentrations of IGF-I before cryopreservation have beneficial effects on sperm functional parameters in rams and pregnancy rates after AI in ewes.

2. Materials and methods 2.1. Animals and semen collection The protocol had been previously approved by the Ethical Committee of the State University of Ceará. This experiment was carried out at the State University of Ceará in the hot semiarid climate of tropical northeast Brazil during the dry season. Five Dorper (meat production breed) 5 rams 24 to 48 mo of age were used. These rams were maintained under a semi-intensive management system, group-housed in a sheltered pen (separate from females), with good quality feed and water available. All rams used had good semen freezability and fertility. Semen collection was done with an artificial vagina (40 – 42 °C) 1 mo before starting the experiment (rams were already accustomed to this procedure). For the experiment, semen from five rams (five ejaculates/ram) was collected once a week and the second ejaculate from each animal was used and treated separately. Immediately after semen collection, ejaculates were kept at 33 °C in a water bath during evaluation. Each ejaculate was evaluated for volume, sperm concentration, mass motility, percentage motile sperm, and vigor, as described [24]. Semen volume was determined using a graduated collection tube, and sperm concentration was determined with a Neubauer’s chamber after dilution to 1:200 in formalin [25]. Mass motility was estimated using a light microscope (Nikon, Tokyo, Japan), using a standardized scale of zero to five [26]. Only ejaculates suitable for freezing (volume ⬎0.5 mL, mass motility ⬎3.5, and sperm concentration ⬎3 ⫻ 109 sperm/mL) [27] were used. 2.2. Experimental design In Experiment 1, a Tris semen extender (3.786 g of Tris) supplemented with 2.11 g citric acid, 0.25 mg mL⫺1 gentamicin, 20% egg yolk, 5% glycerol, and 100 mL distilled water [28] was used. For semen cryopreservation, three concentrations of IGF-I were tested, resulting in four experimental groups: Tris alone (control group) or with IGF-I (Human IGF-I; PeproTech, Inc., Rocky Hill, NJ, USA) at concentrations of 50 (IGF-I 50 group), 100 (IGF-I 100 group) or 250 ng/mL (IGF-I 250 group). The concentrations of IGF-I were chosen based on a previous study performed in other species [29]. In Experiment 2, multiparous crossbred ewes (n ⫽ 121; 1–3 yrs old) were used for AI and randomly assigned to one of two treatments: Tris (control group: 60 ewes) or IGF-I 100 (treatment group: 61 ewes).

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All chemicals were obtained from Sigma, Chemical Co. (St. Louis, MO, USA) unless otherwise stated. 2.3. Exposure and freeze–thaw procedures After semen evaluation, each ejaculate was divided equally into the different groups, packaged into 0.25-mL straws (100 ⫻ 106 sperm/mL) and frozen using a programmable freezer (Freeze Control, CryoLogic, Pty Ltd., Waverley, Australia) with the following freezing curve: cooled at 0.25 °C/min from 33 to 5 °C, kept at this temperature for 120 min and submitted to freezing at 20 °C/min from 5 to ⫺120 °C [30]. Afterward, samples were plunged immediately into liquid nitrogen (⫺196 °C) and stored for 1 mo. One straw from each batch from each ram was thawed, and only batches with ⱖ50% progressive motility were used. The samples were thawed for 20 s in a 40 °C water bath and then evaluated. 2.4. Evaluation of sperm samples 2.4.1. Thermal resistance test After straws from each treatment were thawed, a thermal-resistance test (TRT) was conducted to determine post-thaw sperm longevity for up to 2 h postincubation at 37 °C. Post-thaw samples were evaluated every 1 h for motility, acrosome integrity and osmotic resistance. Sperm motility was evaluated using an aliquot of semen diluted in a sodium citrate solution (2.9%), placed under a coverslip on a glass slide, and evaluated under a phase-contrast microscope (100⫻ magnification) equipped with a heated plate. 2.4.2. Acrosome integrity (NAR) The percentage of sperm with a normal acrosome (NAR) was measured according to Award and Graham [31]. Briefly, 10 ␮L of semen was diluted (1:100) in a saline solution (NaCl 0.9%) with 2% glutaraldehyde, and the proportion of sperm with a non-reacted and normal acrosome was determined under a phase-contrast microscope (1000⫻ magnification). 2.4.3. Osmotic resistance test (HOST) The osmotic resistance test was performed according to Paulenz, et al. [27]. Briefly, an aliquot of semen (25 ␮L) was placed into 200 ␮L of a hypo-osmotic solution (9.0 g fructose, 4.9 g trisodium citrate, distilled water to 1000 mL, 100 mOsm) and was incubated for 45 min at 37 °C. Afterward, 300 ␮L of a 2% glutaraldehyde solution in a cacodylate buffer was added and the percentage of sperm with a swollen tail was recorded. A total of 100 cells from each sample were

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evaluated under a phase-contrast microscope (400⫻ magnification). 2.4.4. Sperm viability Sperm viability was assessed only at 0 h post-thaw. For viability analysis, 300 sperm from each group were analyzed by fluorescence microscopy. Immediately after thawing, one straw from each treatment was evaluated. Semen was incubated in 50-␮L droplets containing 4 ␮M calcein-AM and 2 ␮M ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany) for 15 min at 37 °C. Afterward, sperm were examined under a fluorescence microscope (Nikon, Eclipse 80i, Tokyo, Japan) for evaluation of live/dead fluorescent staining [32]. The emitted fluorescent signals of calcein-AM and ethidium homodimer-1 were collected at 488 and 568 nm, respectively. Sperm were considered alive if stained positively with calcein-AM (green color), and dead if the chromatin was labeled with ethidium homodimer-1 (red color). 2.5. AI fertility trial 2.5.1. Animals and insemination procedures In Experiment 2, AI was performed to test the effect of IGF-I on fertility. Since the IGF-I 100 group had the best motility after 1 and 2 h of incubation, that concentration was selected for use in the AI trials. Multiparous crossbred ewes (n ⫽ 121; 1–3 yrs old) were used and randomly assigned to one of two treatments: Tris (control group: 60 ewes) or IGF-I 100 (treatment group: 61 ewes). Ewes were fed 0.4 kg of concentrate daily and good quality hay and water were available ad libitum. Before insemination, females were synchronized by hormonal treatment as described below. All females were examined with transrectal ultrasonography (5.0 MHz) before synchronization treatment, and those diagnosed as pregnant or with reproductive disorders were excluded. The estrus synchronization protocol used was adapted from Boscos, et al. [33]. Ewes were treated during midbreeding season with 60 mg MAP intravaginal sponges (Progespon, Syntex, Buenos Aires, Argentina) for 12 days and given a 50 ␮g cloprostenol im (PGF2a analog, Ciosin, Coopers, São Paulo, SP, Brazil), along with 400IU of eCG (Novormon, Intervet, São Paulo, SP, Brazil) at sponge removal. Before intrauterine insemination, feed and water were withheld for 24 h. All ewes received 0.5 mL acepromazine im (Acepran 1%, Vetnil, São Paulo, SP, Brazil) as a sedative approximately 45 min before insemination. The time-fixed AI (TFAI) via laparoscopy was performed

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Table 1 Percentages (mean ⫾ SEM) for subjective motility, acrosome integrity (NAR) and hypo-osmotic test (HOST) in ram semen. Treatments (n ⫽ 5 rams)

Endpoint Motility Time 0

Time 1

Tris

64.4 ⫾ 0.8Aa

IGF I 50 IGF I 100 IGFI 250

66.2 ⫾ 0.8Aa 76.2 ⫾ 0.6Ba 74.4 ⫾ 0.5Ba

54.7 ⫾ 0.9Ab 47.2 ⫾ 1.1Ac 56.2 ⫾ 0.9Ab 67.0 ⫾ 0.9Bb 63.6 ⫾ 0.8Cb

Acrosome integrity Time 2

47.9 ⫾ 0.9Ac 58.3 ⫾ 0.9Bc 55.8 ⫾ 0.9Cc

Hypo-osmotic test

Time 0

Time 1

Time 2

Time 0

Time 1

Time 2

84.0 ⫾ 0.7a

61.8 ⫾ 0.6b

51.8 ⫾ 0.6b

50.3 ⫾ 0.3a

32.7 ⫾ 0.4b

22.8 ⫾ 0.3b

82.8 ⫾ 0.5a 84.8 ⫾ 0.6a 85.2 ⫾ 0.7a

66.1 ⫾ 1.9b 66.1 ⫾ 1.9b 67.6 ⫾ 2.2b

50.0 ⫾ 1.6c 49.8 ⫾ 1.8c 49.9 ⫾ 1.6c

50.4 ⫾ 0.2a 51.0 ⫾ 0.4a 50.6 ⫾ 0.3a

34.4 ⫾ 1.5b 35.2 ⫾ 2.0b 33.9 ⫾ 1.8b

23.7 ⫾ 0.3c 24.1 ⫾ 0.4c 24.6 ⫾ 0.7c

Five ejaculates were used for each ram. A-C At each time of evaluation, treatments without a common superscript differed (P ⬍ 0.05). a-c within each treatment, time of evaluations without a common superscript differed (P ⬍ 0.05).

55 h after sponge [34]. All treated ewes were inseminated with 0.2 mL of diluted semen (400 x l06 spz/mL). 2.5.2. Pregnancy assessment Pregnancy diagnosis was performed by transrectal ultrasound (Chison 500vet, Mainland, China, 5.0 MHz) 45 days after AI. 2.6. Statistical analysis Data not normally distributed (according to ShapiroWilk tests), were ranked. Data for sperm motility and acrosome and plasma membrane integrities were analyzed with a SAS MIXED procedure with a repeated statement to account for autocorrelation between sequential measurements (SAS Version 9.2; SAS Institute, Cary, NC, USA). If the effect of group or the interaction of group and time were significant, data were examined further by Duncan’s multiple range test. Data for sperm viability (evaluated by fluorescence microscopy) were analyzed by two-way ANOVA. If the main effect of group was significant, differences among groups were examined by Duncan’s multiple range test. Pregnancy rates were analyzed by ␹2 test of independence. For all statistical analyses, P ⬍ 0.05 was deemed significant. Data are given as the mean ⫾ SEM, unless otherwise stated. 3. Results 3.1. Experiment 1 3.1.1. Effect of IGF-I on acrosome integrity (NAR) and HOST The effects of IGF-I and incubation time (0, 1 and 2 h) after semen thawing on acrosome integrity and hypoosmotic resistance are shown (Table 1). Incubation of semen progressively affected (P ⬍ 0.01) all sperm parameters evaluated (motility, NAR and HOST). However, supplementation with IGF-I in fro-

zen semen, in all concentrations tested in this study (50, 100 and 250 ng/mL) did not affect (P ⬎ 0.05) acrosome integrity or the hypo-osmotic test. 3.1.2. Effect of IGF-I on motility Sperm motility was affected (P ⬍ 0.05) by IGF-I concentration (Table 1). Semen frozen with supplemental IGF-I had higher motility (P ⬍ 0.05) at 0 h (76.2 ⫾ 0.6 and 74.4 ⫾ 0.5% for 100 and 250 ng/mL, respectively) in comparison with the other groups (IGF-I 50: 66.2 ⫾ 0.8 and Tris: 64.4 ⫾ 0.8). After 1 and 2 h post-incubation, motility was highest (P ⬍ 0.05) in the IGF-I 100 group (67.0 ⫾ 0.9 and 58.3 ⫾ 0.9, respectively). 3.1.3. Effect of IGF-I on sperm viability The percentages of viable semen at 0 h were higher (P ⬍ 0.0001) in semen frozen with supplementation of IGF-I at 100 and 250 ng/mL (88.7 and 88.2%) than in the other groups (IGF-I 50: 77.6 and Tris: 76.5%). There is no differences (P ⬎ 0.05) between IGF-I 100 and IGF-I 250 groups. Results of fluorescence analysis from the Tris and IGF-I 100 groups are shown (Fig. 1A, 1B, respectively). 3.2. Experiment 2 Of the 60 ewes inseminated in the Tris group, 38 were pregnant 45 days after FTAI, whereas of 61 ewes inseminated in the IGF-I 100 group, 45 were pregnant. Although the IGF-I group showed a numerically higher pregnancy rate, no difference (P ⬎ 0.05) was observed between ewes inseminated with semen containing Tris alone or IGF-I 100 (63.3 and 73.7%, respectively). 4. Discussion The present study was, apparently, the first to determine the effect of IGF-I on the functional parameters and fertility of ovine frozen-thawed semen. In previous

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A

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B

Fig. 1. Viability assessment of ovine sperm using fluorescent probes. (A) Viable sperm from the IGF-I 100 treatment stained positively with calcein-AM (green fluorescence). (B) Viable and non-viable sperm from the Tris treatment stained positively with calcein-AM (green fluorescence) and labeled with ethidium homodimer -1 (red fluorescence).

studies, IGF-I had pronounced effects on male and female reproductive tracts [18]. In this experiment, three concentrations of IGF-I were tested. For most parameters evaluated, 100 ng/mL was the optimal concentration. Mean concentration of IGF-I in buffalo seminal plasma was 117 ng/mL [29], with similar concentrations of IGF-I in bovine [35] and human seminal plasma [36]. It is well known that IGF-I is a member of the IGF family system, which is composed of IGF-I and IGF-II, two types of receptors (IGFR-1 and IGFR-2), and six binding proteins (IGFBP-1, -2, -3, -4, -5, and -6). The IGFBPs are present in biological fluids and act by inhibiting or potentializing the action of IGFs in target cells [37]. The presence of IGF-I, IGFBP-2, IGFBP-3, and IGFBP-4, as well as prostate-specific antigen in human seminal plasma, suggests a complex interaction that may regulate sperm physiology and activity [38]. Circulating IGF-I is mainly produced by the liver, but most other cell types can also produce IGF-I, which acts in an autocrine/paracrine manner [39]. In humans, the binding receptor for IGF-I has been localized on Leydig cells [40], spermatocytes, spermatids [41] and Sertoli cells, the main source of testicular IGF-I [42,43]. The effects of IGF-I, characterized by higher rates of sperm motility and viability in the present study, was associated with a significant increase in total motility, progressive forward motility, functional membrane integrity, and a decrease in lipid peroxidation during cryopreservation of buffalo semen [29]. Moreover, some studies demonstrated the positive effect of IGF-I on sperm functions, such as motility and viability, in rabbits and bulls [22,23]. The cooling process alters the fluidity of the phospholipid bilayer, making the phospholipids cluster and exclude membrane

proteins, reducing their mobility and thus the membrane functionality [44]. In a recent study, the addition of insulin to the extender increased motility, kinematics and acrosome integrity of frozen sheep semen [45]. Insulin may promote greater glucose uptake by sperm through replacement of glucose transport proteins, which are probably lost during the cooling process, providing more substrate for maintenance of flagellar activity. Other growth factors, including EGF and TGF␣, have been reported in seminal plasma [46,47]. These substances, in association with additional growth factors and hormones in seminal plasma, suggest a regulatory role of growth factors in sperm regulation processes. In this experiment, gentamycin, a well-known mitochondrial ribosomal inhibitor [48] was present in the semen extender. Furthermore, IGF-I protects cells from hypoxia/reoxygenation injury via stabilizing mitochondria and reducing reactive oxidative species damage [49]. We inferred that these two compounds, acting together, accounted for the positive results in the present study. Although the current study provided evidence that IGF-I improved some functional parameters of semen in vitro, beneficial effects were not observed on fertility (pregnancy rate), even though the number of pregnant females inseminated with semen cryopreserved with Tris extender containing IGF-I at 100 ng/mL was numerically higher than that with Tris alone. Thus, further studies are necessary to understand the metabolic mechanism by which IGF-I acts and its effects on fertility. In conclusion, supplementation of Tris extender with IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after lapa-

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roscopic insemination of ewes with frozen-thawed semen.

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Acknowledgments Rodrigo Tenório Padilha is the recipient of a grant from FACEPE, Recife, PE, Brazil.

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