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Fırat Üniversitesi Sağlık Bilimleri Veteriner Dergisi
2015, Cilt 29, Sayı 3, Sayfa(lar) 183-186
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Ovsynch Senkronizasyonundan Hemen Önce Vitamin E ve Selenyum Enjeksiyonunun Sütçü İneklerde Gebe Kalma Oranı, Antioksidant Aktivite ve Progesteron Düzeyleri Üzerine Etkisi
Atilla YILDIZ1, Engin BALIKÇI2, Fuat GÜRDOĞAN1
1Fırat Üniversitesi, Sivrice Meslek Yüksekokulu, Elazığ, TÜRKİYE
2Fırat Üniversitesi, Veteriner Fakültesi, İç Hastalıkları Anabilim Dalı, Elazığ, TÜRKİYE
Anahtar Kelimeler: Konsepsiyon, glutatyon peroksidaz, östrus senkronizasyonu, süperoksid dismutaz
Özet
Bu çalışmanın amacı, ovsynch senkronizasyonundan hemen önce tek doz derialtı uygulanan vitamin E ve selenyumun, laktasyondaki sütçü ineklerin gebe kalma oranları, antioksidan aktiviteleri ve plazma progesteron düzeyleri üzerine etkisini araştırmaktı. Yirmi dört sağlıklı inek rastgele deney (grup E, n=14) ve kontrol (grup C, n=10) grubuna ayrıldı ve ovsynch protokolüyle senkronize edildi. Grup E'de ineklere ovsynch senkronizasyonundan hemen önce tek doz 8 mL Esegal (60 mg/mL vitamin E and 1 mg/mL sodium selenite) derialtı yolla verildi. Grup C'deki ineklere uygulama yapılmadı. Her iki gruptaki ineklerden tohumlamadan hemen önce ve 14 gün sonra kan örnekleri alındı. Gebelik teşhisi tohumlamadan 60 gün sonra yapıldı. Gruplar arasında gebe kalma oranlarında önemli farklılık bulunmadı. Tohumlamadan sonraki 14. günde grup E'de ortalama glutathione peroxidase (GSH-Px) ve superoxide dismutase (SOD) düzeylerinin grup C'dekine göre ve uygulama öncesi kendi düzeylerinden daha yüksek olduğu (P<0.05) tespit edildi. Progesteron düzeyleri tohumlamadan sonraki 14. günde grup C ile karşılaştırıldığında grup E'de arttığı belirlendi. Sonuç olarak, ovsynch'ten hemen önce tek doz derialtı uygulanan vitamin E ve selenyumun gebe kalma oranı üzerinde etkili olmadığı görüldü. Bununla birlikte, uygulamanın eritrosit GSH-Px ve SOD aktiviteleri ve plazma progesteron düzeyleri üzerine olumlu bir etkisi tespit edildi.
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    Oxidative stress is one of the major causes of the decrease in viability and development of embryo1. Embryo is highly sensitive to injury to oxidant molecules because of its low antioxidant capacity2. In the various researchs, studies focused on studying antioxidant strategies to prevent oxidative stress induced damage to gametes and developing embryos3. It is known that vitamin E (vit E) and selenium (Se) are powerful free-radical scavenger and antioxidants. Vit E prevents the oxidative chain from proliferating prior to the alteration of neighboring molecules by being oxidized into harmless compounds4,5. Selenium is important in inhibiting nitric oxide production in cells6 and is an essential element of several enzyme systems, including glutathione peroxidase (GSH-Px) and deiodinase7. These enzymes have been shown to be important in embryonic development, through their antioxidant functions and regulatory effects on metabolic rate8. In addition to, plasma GSH-Px activity is assumed to be indicative of oxidative stress9. Vit E and Se are capable of improving the developmental capacity of bovine embryos in vitro10,11. Vit E and Se may suppress these oxidants. Several studies have shown that administration of Se, vit E or vit E and Se in combination resulted in higher pregnancy rates12-14. However, other studies have shown little or no beneficial effect of administration of vit E and/or Se15-17. Oxidative stress and maternal low blood progesterone levels during early embryonic development are a major cause of decreased pregnancy rate in dairy cows. Vierk et al.18 suggested that luteolysis of the corpus luteum is due to accumulation of oxidative species. Circulating progesterone concentrations are critical for maintenance of pregnancy. It has been suggested that some of early pregnancy losses in livestock may be due to insufficient circulating progesterone concentrations19. Hence, use of appropriate antioxidants can help to improve conception rate by preventing accumulation of oxidative species during early embryonic development in dairy cows. However, whether vit E and Se posses similar benefits on the conception rate in cows has yet to be determined.

    The objective of this study was to investigate the effect of a single subcutaneous injection of vit E and Se administered just before ovsynch on erythrocyte GSH-Px and superoxide dismutase (SOD) activities and plasma progesterone levels and pregnancy rates of lactating dairy cows.
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    The study was carried out in a conventional dairy farm located in Elazig (Eastern Turkey). A total of 24 healthy multiparous Holstein cows, aged 4-7 years, and with mean body condition score of 2.74±0.17 (on scale 1 to 5)20 were used in this study in the period of February–April. The cows were in 55–70 days postpartum. They were all examined by rectal palpation and vaginoscopic examination to evaluate the normality of their reproductive tract. They had not any disorders in their reproductive tracts. The cows were fed a total mixed ration (TMR) of corn silage and grains that was balanced according to nutritional requirements based on milk production, and housed in a tie-stall confinement facility. Ovsynch program was performed with 2 injections of GnRH (10 μg Busereline acetate, Receptal®, Intervet Istanbul, Turkey), 7 d before and 48 h after an injection of PGF2 alfa (25 mg, Dinoprost tromethamine, Dinolytic®, Etkin, Istanbul, Turkey). The cows were divided randomly into two groups. The experimental group (group E) (n= 14) was administered a single subcutaneous injection of 8 mL Esegal per animal (Esegal inj.: 60 mg vitamin E and 1 mg sodium selenite in 1 mL, Galenka) just before the first GnRH injection and the control group (group C) was not received vit E and Se (n = 10). Cows were artificially inseminated 16 h after the second injection of Gonadotrophin releasing hormone (GnRH). Pregnancy was determined by palpation per rectum at 60 d after AI.

    Blood samples were taken from jugular vein into tubes containing EDTA immediately before treatment and on d 14 after AI for cows per group, to monitor erythrocyte GSH-Px, SOD activities and plasma progesterone levels.

    Erythrocyte GSH-Px enzyme activity was determined using the method previously described in Paglia and Valentine,21 and was expressed as Units/gram of Hemoglobin (U/g Hb).

    Hemoglobin (U/g Hb). Erythrocyte SOD activity was measured at 560 nm and was expressed as U/gHb22.

    Plasma progesterone level was quantified by a commercial solid-phase RIA kits (Coat-a-count; Diagnostic Products Corp., Los Angeles, CA, USA)23. The assay sensitivity was 0.05 ng/mL. The intra– and inter– assay coefficients of variation were 7.6 and 7.9% respectively.

    The pregnancy rate was compared between groups by Fisher's exact test. Progesteron levels, GSH-Px and SOD activities means between groups were tested using student T test, whereas pre and post–treatment means of GSH-Px and SOD activities were compared using paired t test. Mean values are presented as mean±standard deviation (SD). Differences were considered as significant at P<0.05.
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    The conception rate for first insemination was 42.9% (6 to 14) in group E, and 30.0% (3 to 10) in group C. Conception rate was proportionately higher in cows of treatment group, compared to cows in control group. However, this increase was not statistically significant at the 5% level (P>0.05). The mean GSH-Px and SOD activities of group E were similar to group C immediately before vit E and Se treatment (Table I).


    Büyütmek İçin Tıklayın    		 Table 1: The mean levels and standard deviations (±SD) of GSH-Px, SOD and Progesterone for group E and group C

    On day 14 after AI, the GSH-Px and SOD activities were higher in group E (P<0.05) than group C. On day 14 after AI, GSH-Px and SOD activities in group C were higher than the initial activities but this increase was not considerable (P>0.05). Whereas, the differences for GSH-Px and SOD activities between immediately before application and on day 14 after AI in the in group E were statistically significant (P<0.05). The progesterone levels increased significantly (P<0.05) in group E when compared with group C on day 14 after AI.
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    The effects of Se and vit E supplementation on conception rates in cattle have been examined in feeding trials, but the effect of Se and vit E on conception rate remains controversial. Many studies have reported to demonstrate a significant difference in conception rates between treatment groups following Se or Se plus vit E supplementation13,24, but this finding was not consistent25,26. In the present study, although conception rate for group E were observed to be proportionately higher than group C, this was not considerable (P>0.05). A similar lack of effect on conception rates was also found by Paula-Lopes et al.26, Aréchiga et al.27 and Awadeh et al.28. However, this is in contrast with the results of Castro et al.13 and Zanella et al.14. The variation in results may all contribute to different study designs and methods, conditions and management in farms, change of diet, breeding of animals, stress factors. Several possible reasons may explain why vit E and Se injection in this study did not improve conception rate in cows. First, the period in which cows injected vit E and Se may not be suitable to prevent effects of oxidative stress during early embryonic development. Also, more complex antioxidant therapies may be necessary to achieve beneficial effects. Finally, antioxidants did not totally prevent effects of reactive oxygen species on embryonic survival.

    On day 14 after AI, the mean GSH-Px activity in treated group were higher (P<0.05) when compared to control group. The same variations were observed in mean GSH-Px activity of group E on day 14 after AI in comparison with the initial level, and these changes were considerable (P<0.05). The mean GSH-Px and SOD activities in group C on day 14 after AI was higher than the initial level, but this increase was not considerable (P>0.05). Thus, this result indicated that vit E and Se injection can responsible for an increase in antioxidant activity. Similar to our results, a significant increase in GSH-Px activity due to Se supplementation was reported in the erythrocytes of cows29,30. GSH-Px has been shown to be important in embryonic development, through their antioxidant functions and regulatory effects on metabolic rate8. In the present study, however, pregnancy rates were not affected by increased GSH-Px and SOD activities.

    In the present study, vit E and Se injection raised plasma progesterone levels. Some antioxidant systems actively work in the corpus luteum during pregnancy31. It is known that vit E and Se is also an antioxidant, and could play a role in the antioxidant system in the corpus luteum32. It is possible that Se degrades H2O2 or peroxides in the corpus luteum as a component of GSH-Px33 or phospholipid hydroperoxide glutathione peroxidise34. This would support its hormone production activity. Kamada and Hodate32 have reported the positive effects of Se on plasma progesterone concentration in the progesterone production of luteal cells.

    In conclusion, a single subcutaneous injection of vitamin E and selenium immediately before ovsynch may not have a significant effect on conception rates of lactating dairy cows. However, this application had a beneficial effect on antioxidant activity and plasma progesterone levels. Vit E and Se could induce increased progesterone secretion by the corpus luteum which is necessary for successful pregnancy by playing a role in the antioxidant system in the corpus luteum. Treatment with vitamin E and Se proportionally increased conception rates of cows in this study. Lack of difference in conception rates in this study was likely due to the use of limited numbers of animals. Therefore, the results highlight the need for additional studies to investigate the beneficial effects of vitamin E and Se on conception rates.
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    1) Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res 2008; 44: 280-287.

    2) Thompson LP, Al-Hasan Y. Impact of oxidative stress in fetal programming. J Pregnancy 2012: 1-8. doi: 10.1155/ 2012/582748

    3) Agarwal A, Gupta S, Sikka S. The role of free radicals and antioxidants in reproduction. Curr Opin Obstet Gynecol 2006; 18: 325-332.

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    6) Faes MR, Caldas-Bussiere MC, Viana KS, et al. Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium. Anim Reprod Sci 2009; 110: 222-236.

    7) Sales JNS, Pereira RVV, Bicalho RC, Baruselli PS. Effect of injectable copper, selenium, zinc and manganese on the pregnancy rate of crossbred heifers (Bos indicus¡ÁBos taurus) synchronized for timed embryo transfer. Livestock Sci 2011; 142: 59-62.

    8) Hostetler CE, Kincaid RL, Mirando MA. The role of essential trace elements in embryonic and fetal development in livestock. The Vet J 2003; 166: 125-139.

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    10) Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR. Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil 1991; 92: 125-131.

    11) Olson SE, Seidel GE Jr. Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients. Biol Reprod 2000; 62: 248-252.

    12) Richardson MJ, Lemenager RP, Pyatt N, Lake SL. Natural source vitamin E supplementation and reproductive efficiency in beef cows. Proc West Sect Amer Soc Anim Sci 2008; 59: 339-342.

    13) Castro CSJ, Guerra LJE, Cordova IA, Soto MLE, Guerra CJE. Pregnancy rate on Ayrshire cows supplemented with selenium and vitamin E. In: Proc. 14th Int Cong, the International Society for Animal Hygiene (ISAH), Vechta, Germany, pp. 193-195, 2009.

    14) Zanella R, Bondan C, Soares JCM, Zanella EL, de Lima MR. Use of antioxidants to improve the pregnancy rate in beef cattle submitted to a synchronization protocol with progesterone (P4). Ci Anim Bras 2010; 11: 477-481.

    15) Spears JW, Harvey RW, Segerson EC. Effects of marginal selenium deficiency and winter protein supplementation on growth, reproduction and selenium status of beef cattle. J Anim Sci 1986; 63: 586-594.

    16) Hidiroglou M, McAllister AJ, Williams CJ. Prepartum supplementation of selenium and vitamin E to dairy cows: assessment of selenium status and reproductive performance. J Dairy Sci 1987; 70: 1281-1288.

    17) Stowe HD, Thomas JW, Johnson T, et al. Responses of dairy cattle to long-term and short-term supplementation with oral selenium and vitamin E. J Dairy Sci 1988; 71: 1830-1839.

    18) Vierk JE, Hansen TR, Austin KJ, et al. Inhibition by tocopherol of prostaglandin-induced apoptosis in ovine corpora lutea. Prostaglandins Other Lipid Mediat 1999; 56: 265-276.

    19) Long NM, Tuersunjiang N, George LA, et al. Maternal nutrient restriction in the ewe from early to midgestation programs reduced steroidogenic enzyme expression and tended to reduce progesterone content of corpora lutea, as well as circulating progesterone in nonpregnant aged female offspring. Reprod Biol Endocrinol 2013; 11: 34.

    20) Edmonson AJ, Lean IJ, Weaver LD, Farver T, Webster G. A body condition scoring chart for Holstein dairy cows. J Dairy Sci 1989; 72: 68-78.

    21) Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxi-dase. J Lab Clin Med 1967; 70: 158-169.

    22) Sun Y, Oberley LW, Ying L. A simple method for clinically assay of superoxide dismutase. Clin Chem 1988; 34: 497-500.

    23) Ginther OJ, Silva LA, Araujo RR, Beg MA. Temporal associations among pulses of 13,14-dihydro-15-keto-PGF2alpha, luteal blood flow, and luteolysis in cattle. Biol Reprod 2007; 76: 506-513.

    24) Allan CL, Hemingway RG, Parkins JJ. Improved reproductive performance in cattle dosed with trace element/vitamin boluses. Vet Rec 1993; 132: 463-464.

    25) Gunter SA, Beck PA, Phillips JM. Effects of supplementary selenium source on the performance and blood measurements in beef cows and their calves. J Anim Sci 2003; 81: 856-864.

    26) Paula-Lopes FF, Al-Katanani YM, Majewski AC, McDowell LR, Hansen PJ. Manipulation of antioxidant status fails to improve fertility of lactating cows or survival of heat-shocked embryos. J Dairy Sci 2003; 86: 2343-2351.

    27) Ar¨¦chiga CF, V¨¢zquez-Flores S, Ort¨ªz O, et al. Effect of injection of ¦Â-carotene or vitamin E and selenium on fertility of lactating dairy cows. Theriogenology 1998; 50: 65-76.

    28) Awadeh FT, Kincaid RL, Johnson KA. Effect of level and source of dietary selenium on concentrations of thyroid hormones and immunoglobulins in beef cows and calves. J Anim Sci 1998; 76: 1204-1215.

    29) Hogan JS, Smith KL, Weiss WP, Todhunter DA, Schockey WL. Relationships among vitamin E, selenium, and bovine blood neutrophils. J Dairy Sci 1990; 73: 2372-2378.

    30) Ortman K, Pehrson B. Effect of selenate as a feed supplement to dairy cows in comparison to selenite and selenium yeast. J Anim Sci 1999; 77: 3365-3370.

    31) Sugino N, Takiguchi S, Kashida S, et al. Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy. Mol Hum Reprod 2000; 6: 19-25.

    32) Kamada H, Hodate K. Effect of dietary selenium supplementation on the plasma progesterone concentration in cows. J Vet Med Sci 1998; 60: 133-135.

    33) Flohe L, G¨¹nzler WA, Schock HH. Glutathione peroxidase: A selenoenzyme. FEBS Letters 1973; 32: 132-134.

    34) Kamada H, Ikumo H. Effect of selenium on cultured bovine luteal cells. Anim Reprod Sci 1997; 46: 203-211.
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