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Cystic ovarian follicles and thyroid activity in the dairy cow
Animal Reproduction Science 138 (2013) 150–154
Contents lists available at SciVerse ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Cystic ovarian follicles and thyroid activity in the dairy cow M. Mutinati, A. Rizzo, R.L. Sciorsci ∗ Department of Emergencies and Organ Transplantation (D.E.T.O.), Faculty of Veterinary Medicine, University of Bari “Aldo Moro”, s.p. per Casamassima, km 3, 70010 Valenzano (Bari), Italy
a r t i c l e
i n f o
Article history: Received 31 October 2012 Received in revised form 20 February 2013 Accepted 28 February 2013 Available online 15 March 2013 Keywords: Dairy cows Cystic ovarian follicles Thyroid hormones
a b s t r a c t Thyroid activity affects the functionality of the reproductive axis and thyroid dysfunction has been associated with ovarian hyperstimulation syndrome and polycystic ovarian syndrome, in human medicine. This study investigates serum17- estradiol, progesterone, thyrotropic and thyroid hormone levels, in cyclic dairy cows on heat (Group H) and in dairy cows with ovarian follicular cysts (Group FC). Both 17- estradiol and progesterone serum concentrations were statistically higher in cystic than in cyclic cows (estradiol: 8.51 ± 1.91 vs 6.32 ± 1 pg/mL) (progesterone: 0.49 ± 0.17 vs 0.13 ± 0.03 ng/mL), whereas TSH and fT4 serum concentrations were statistically lower in cows with cystic ovarian follicles (COF), compared to cyclic ones (TSH: 2.48 ± 1.31 vs 3.56 ± 1.03 ng/mL) (fT4: 5.86 ± 1.69 vs 8.63 ± 1.08). fT3 serum levels were similar, in both cystic and cyclic subjects (2.94 ± 0.65 vs 3.02 ± 0.9, respectively). Based on these results it was decided to examine the function of the thyrothropic axis of dairy cows in a similar manner to that conducted on humans. If severe hypothyroidism should be found, a hormone replacement therapy could be attempted in cystic cows refractory to “ordinary” therapies. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cystic ovarian follicles (COF) represent one of the most common reproductive disorders affecting dairy herd fertility, occurring with an incidence of 6–29%, mainly in the postpartum of dairy cows (Silvia et al., 2005; Vanholder et al., 2006; Grado-Ahuir et al., 2011). According to some authors follicular cysts can be defined as follicle-like structures, with an ovulatory size, persisting for more than 6 days without luteinizing. These structures are likely to undergo spontaneous regression, within the first 50 days postpartum, otherwise they persist or regress being later replaced by another cyst on the same or opposite ovary (Silvia et al., 2005; Rizzo et al., 2009a; Rizzo et al., 2011a).
∗ Corresponding author. Tel.: +39 0805443881; fax: +39 0805443883. E-mail address: [email protected] (R.L. Sciorsci). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.02.024
A large number of factors can influence the development of follicular cysts in dairy cows, in primis an alteration of the hypothalamo-hypophyseal-gonadal axis (Vanholder et al., 2006; Roth et al., 2012), is probably due to an alteration in the feedback mechanism of 17- estradiol on the hypothalamus or to a desensitization of hypothalamic GnRH and ovarian LH receptors by sustained 17- estradiol and LH concentrations, respectively (Sciorsci et al., 2003). Asynchrony between LH surge and up-regulation of LH receptors on the follicular wall seems to play an important role as a cofactor in follicular cyst formation (Vanholder et al., 2006). Vascular dysfunction (Rizzo et al., 2009a), an unbalance between reactive oxygen species (ROS) and antioxidants (Rizzo et al., 2009b), alterations in ovarian sympathetic nerve activity (Rizzo et al., 2011b), severe negative energy balance (NEB) during postpartum, with consequent reduction in peripheral plasma concentrations of insulin-like growth factors (IGFs), insulin, glucose and leptin (Beam and Butler, 1999; Lucy, 2003; Block et al., 2011), changes in the
M. Mutinati et al. / Animal Reproduction Science 138 (2013) 150–154
expression of heat shock proteins (hsp) in the granulosa and theca cells of the follicular wall (Velázquez et al., 2011) are also contributing factors, to the onset of cystic ovarian follicles, in the dairy cow. In human medicine, thyroid disorders have been associated with ovarian hyperstimulation syndrome (OHSS) (Vasseur et al., 2003; Montanelli et al., 2004; Shu et al., 2011) and with polycystic ovary syndrome (PCOS) (Jung et al., 2011). Both hypothyroidism (Shu et al., 2011) and hyperthyroidism (Jung et al., 2011) have been associated with ovarian cysts in pre-menopausal women. In hypothyroid women developing ovarian cysts, hormone replacement therapy results in a restoration of the euthyroid state as well as a regression of symptoms and ovarian enlargement (Shu et al., 2011). In contrast, Jung et al. (2011) reported that treatment with propylthiouracil is effective in restoring fertility, physiological ovarian appearance and euthyroid function in women with Grave’s hyperthyroidism and PCOS. Therefore this study aimed to investigating steroid, thyrotropic hormone (TSH) and free thyroid hormone (fT3 and fT4) blood concentrations in healthy and infertile dairy cows diagnosed with ovarian follicular cysts.
2. Material and methods In this study all procedures were conducted in accordance with the EU Directive 2010/63/EU. The study was performed between February and July 2012, at a commercial dairy farm in the prefecture of Taranto (Apulian Region, South Italy), consisting of 280 Holstein Friesian cows kept in a semi-intensive regimen and fed pasture, hay, minerals and concentrates; fresh water was available ad libitum. Two clinical examinations were performed 10 days apart using transrectal palpation and ultrasonography (SonoSite, MicroMaxx Bothell, WA, USA, linear probe 5–10 MHz, set at 7.5 MHz). This was used to diagnose cystic ovarian follicles in 30 cows, with a total of 60 cows (including those with CF) chosen and enrolled in the study. The diagnosis of ovarian follicular cysts was performed ultrasonographically, keeping into account the criteria indicated by Silvia et al. (2005), i.e.: mean diameter of at least 17 mm, wall thickness <3 mm, blood progesterone <1 ng/mL, persistence for more than 6 days. The 60 cows chosen, free from common parasites and infective agents, were between the second and third month of lactation (60 ± 10 days post partum) and between the third and the fifth lactation (27 kg/d) and had a mean BCS of 3 (Roche et al., 2009) The choice of cows being in the same, the narrow phase of lactation was chosen as thyroid activity is known change with stage of lactation (Nixon et al., 1988). The cows were divided into two groups of 30 subjects each:
Group CF: cows with ovarian follicular cysts Group H: healthy cows on heat.
151
2.1. Blood samples Blood samples were taken from the coccygeal vein, using vacutainer serum tubes. These were kept on ice and taken to the lab in a mean time of 50 ± 20 min. The blood samples employed for this experimentation were the same used for endocrine routine screening, performed to assess the cystic or cyclic heat condition respectively, and were collected at the following times: • on the day in which ovarian follicular cysts were diagnosed (Group CF); • on the day of heat (Group H). Blood collection was performed at 5:00 pm, in order to avoid the interference of the circadian rhythm on thyroid hormone levels (Russell et al., 2008). Once in the lab, blood samples were centrifuged at 1620 × g for 10 min at +4 ◦ C and sera were stored in eppendorf (1.5 mL) at −20 ◦ C, until analytical determinations. On serum, 17- estradiol, progesterone, TSH, fT3 and fT4 concentrations were assessed. An ELISA kit was employed for the assessment of all these parameters. Particularly, 17- estradiol high sensitivity ELISA kit (sensitivity 14 pg/mL; specificity 100%) and progesterone ELISA kit (sensitivity 8.57 pg/mL; specificity 100%) (Enzo® Life Sciences, Postfach, Switzerland) were used for dosing 17- estradiol and progesterone. Bovine TSH was dosed with a quantitative ELISA Test kit (Bovine thyroid stimulating hormone elisa test kit end-erkb1015) (Vinci Biochem, Vinci, Florence, Italy), sensitivity: 0.2 ng/mL; specificity: 100%. ELISA kits (fT3, Cat 1650; fT4 Cat 1110, Alpha Diagnostic International, San Antonio, Texas, USA), sensitivity 0.3 pg/mL and 1 pg/mL for ft3 and ft4, respectively; specificity 100% (fT3 and fT4) were used for the determination of serum fT3 and fT4 concentrations.
2.2. Statistical analysis Statistical analysis was conducted using IBM SPSS Statistics 19 (IBM Corporation Software group, Somers, NY, USA). Student’s T test for independent variables was used for intergroup comparison, whereas Person’s test was employed for correlations among 17- estradiol, progesterone, TSH, fT3 and fT4, in both groups and a value of P < 0.05 was set as significance level.
3. Results The data obtained (Mean ± s.d.) are shown in Figs. 1–5. 17- estradiol and progesterone serum concentrations in cyclic cows on heat and in cystic ones were similar to those reported in literature (Bottarelli, 1989; Lucy, 2003; Todoroki et al., 2011; Roth et al., 2012). Both 17 estradiol and progesterone serum concentrations were statistically higher in the cows with follicular cysts
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Fig. 1. Serum 17- estradiol concentrations (pg/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.01. Fig. 4. Serum fT3 concentrations (pg/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC).
Fig. 2. Serum Progesterone concentrations (ng/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.001. Fig. 5. Serum fT4 concentrations (pg/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.01.
(1988) and Nixon et al. (1988), whereas fT4 was found to be a little lower than its physiological levels (Anderson et al., 1988; Nixon et al., 1988). TSH and fT4 serum concentrations were lower in Group FC than in Group H, with TSH and fT4 levels giving rise to statistically significant differences (p < 0.05 and p < 0.01, respectively). Pearson’s test revealed no significant correlations, among the parameters tested, except for TSH and fT3 serum concentrations which were negatively and significantly correlated, in Group H (p < 0.05; R = −0.567). Fig. 3. Serum TSH concentrations (ng/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.001.
(Group FC), compared to healthy ones (Group H) (p < 0.01 and p < 0.001, respectively) and confirms what has been previously reported in the literature (Sciorsci et al., 2003). In both groups, serum TSH concentrations were comparable to those found by De Moraes et al. (1998), fT3 concentrations resembled those found by Anderson et al.
4. Discussion and conclusions In the present study, the endocrine milieu in cyclic and cystic dairy cows was investigated. Particular attention was focused on thyroid hormones, since, to the best of our knowledge, only uncertain and old reports dealing with the interaction of thyroid hormones and the establishment of cystic ovarian follicles in the dairy cow, exist (Dzhambazov, 1976).
M. Mutinati et al. / Animal Reproduction Science 138 (2013) 150–154
In human reproduction, numerous studies have been performed on the interaction among thyroid diseases, infertility and ovarian dysfunctions (Jung et al., 2011; Shu et al., 2011). Furthermore, a strong interaction between thyroid and ovary is inferred from many in vitro researches, both in humans and animals. In fact, the presence of Sodium-Iodide Symporter (NIS) in human and murine ovaries, that of triiodothyronine (T3) and thyroxine (T4) in the ovarian follicular fluid of farm animals, of 5 deiodinase and of thyroid hormone receptors in oocytes, cumuli and porcine and human granulosa cells (Slebodzinski, 2005) have been found. In another study, thyroglobulin (TBG) and TSH receptor were detected in bovine luteal cells by immunohistochemistry (Mutinati et al., 2010). Besides the detection of factors implied in thyroid hormone synthesis and activity, other in vitro studies confirmed a direct role of T3 and T4 in promoting steroidogenesis in human, porcine and bovine follicular and luteal cells (Gregoraszczuk and Galas, 1998; Spicer et al., 2001). In the present workserum fT4 concentrations were statistically lower, in cystic cows, than in cyclic ones, whereas fT3 serum levels were similar in both groups. Free T4 is the major secretory product within the thyrocite, whereas fT3 circulating levels mainly derive from the activity of 5 deiodinase, in peripheral tissues (Kelly, 2000). Given these observations, it is likely that the statistically significant decrease of circulating fT4 found in the cystic cows is due either to a decreased thyroid activity or to an increased activity of peripheral deiodinases. The in vitro studies of Gregoraszczuk and Galas (1998) and Gregoraszczuk (2000) showed that T3 elicits progesterone and androgen secretion in porcine luteal and thecal cells and up-regulation of estrogen receptors in granulose cells. Later, Spicer et al. (2001) demonstrated that T3 is able to enhance aromatase activity in cultured bovine granulose cells, in presence of insulin and FSH. It is well known from literature that cystic cows display higher circulating estradiol and progesterone levels, compared to cyclic ones, according to what occurred in this study (Todoroki et al., 2011; Roth et al., 2012). Given the effects of T3 on steroidogenesis, an involvement of this hormone in the high 17- estradiol and progesterone levels detected in Group FC cannot be excluded. In fact, fT3 may synergistically act with the sustained blood LH levels, which are detectable in cows with ovarian cysts (Hamilton et al., 1995). The higher estrogen blood concentrations found in cystic cows compared to cyclic subjects, in turn, could contribute to a reduction in TSH pituitary synthesis, given the strict relationship between estrogens and the hypothalamic-pituitary-thyroid axis inferred by Léan et al. (1977) and later confirmed by Böttner et al. (2006). In this study, TSH concentrations found in cystic cows are lower than those found in cyclic ones and this may be due to a desensitizing effect exerted by the high blood 17- estradiol detected in the same group, according to the direct interactions between estrogens and TRHinduced TSH secretion (Böttner et al., 2006). However, the decreased serum TSH levels are still within the physiological ranges, in both groups (De Moraes et al., 1998) and this accounts for the physiological levels of
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fT3 detected (Nixon et al., 1988), despite the decrease in fT4 serum concentrations found in the cystic group. The reduction in the blood fT4 levels detected in this study may have depended both on a reduction in the thyrotropic activity of TSH and on an increase in peripheral deiodinase activity, which is known to be regulated by TSH (Kelly, 2000). In conclusion, the results of this study confirm that, as in human medicine, also in the bovine species ovarian diseases may be associated with alterations in thyroid activity, but whether thyroid dysfunction is a cause involved in the onset of ovarian cysts, (Jung et al., 2011; Shu et al., 2011) or it is a consequence of the sustained steroid hormone concentration accompanying the cystic condition, is still a matter of debate. Given these results, a thyroid hormone screening could be useful in those cases of CF refractory to common therapies. In this investigation, a slight decrease in fT4 circulating levels, with no effect on fT3 blood concentrations was detected. Should fT3 be lower than its physiologic levels (Anderson et al., 1988; Nixon et al., 1988), a therapeutic approach based on l-thyroxine administration could be attempted, as it routinely occurs in human medicine.
References Anderson, R.R., Nixon, D.A., Akasha, M.A., 1988. Total and free thyroxine and triiodothyronine in blood serum of mammals. Comp. Biochem. Physiol. 89 (3), 401–404. Beam, S.W., Butler, W.R., 1999. Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. J. Reprod. Fertil. 54, 411–424. Block, S.S., Butler, W.R., Ehrhardt, R.A., Bell, A.W., Van Amburgh, M.E., Boisclair, Y.R., 2011. Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance. J. Endocrinol. 171, 339–348. Bottarelli, F., 1989. Fertilità ed Ipofertilità Bovina. Ed. Tep Veterinaria, Castelgiovanni, Italy. Böttner, M., Christoffel, J., Rimoldi, G., Wuttke, W., 2006. Effects of longterm treatment with resveratrol and subcutaneous and oral estradiol administration on the pituitary-thyroid-axis. Exp. Clin. Endocrinol. Diabetes 114 (2), 82–90. De Moraes, G.V., Vera-Avila, H.R., Lewis, A.W., Koch, J.W., Neuendorff, D.A., Hallford, D.M., Reeves, J.J., Randel, R.D., 1998. Influence of hypo- or hyperthyroidism on ovarian function in Brahman cows. J. Anim. Sci. 76, 871–879. Dzhambazov, G.P., 1976. Cyst formation in the ovaries of cows. Vet. Med. Nauki 13 (2), 83–92. Grado-Ahuir, J.A., Aad, P.Y., Spicer, L.J., 2011. New insights into the pathogenesis of cystic follicles in cattle: microarray analysis of gene expression in granulose cells. J. Anim. Sci. 89, 1769–1786. Gregoraszczuk, E.L., 2000. Is thyroid hormone a modulator of estrogen receptor in porcine follicular cells? Endocr. Regul. 34 (3), 151–155. Gregoraszczuk, E.L., Galas, J., 1998. In vitro effect of triiodothyronine on the cyclic AMP, progesterone and testosterone level in porcine theca, granulose and luteal cells. Endocr. Regul. 32 (2), 93–98. Hamilton, S.A., Garverick, H.A., Keisler, D.H., Xu, Z.Z., Loos, K., Youngquist, R.S., Salfen, B.E., 1995. Characterization of ovarian follicular cysts and associated endocrine profiles in dairy cows. Biol. Reprod. 53 (4), 890–898. Jung, H.J., Hahm, J.R., Jung, T.S., Kim, H.J., Kim, H.S., Kim, S., Kim, S.K., Lee, S.M., Kim, D.R., Choi, W.J., Seo, Y.M., Chung, S.I., 2011. A 27-yearold woman diagnosed as polycystic ovary syndrome associated with Graves’ disease. Int. Med., doi:10.2169/internalmedicine.50.5475. Kelly, G., 2000. Peripheral metabolism of thyroid hormones: a review. Altern. Med. Rev. 5 (4), 306–332. Léan, A.D., Ferland, L., Drouin, J., Kelly, P.A., Labrie, F., 1977. Modulation of pituitary thyrotropin releasing hormone receptor levels by estrogens and thyroid hormones. Endocrinology 100 (6), 1496–1504. Lucy, M.C., 2003. Mechanisms linking nutrition and reproduction in postpartum cows. Reproduction 61, 415–427.
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Montanelli, L., Delbaere, A., Di Carlo, C., Nappi, C., Smits, G., Vassart, G., Costagliola, S.A., 2004. Mutation in the follicle-stimulating hormone receptor as a cause of familial spontaneous ovarian hyperstimulation syndrome. J. Clin. Endocrinol. Metab. 89 (3), 1255–1258. Mutinati, M., Desantis, S., Rizzo, A., Ventriglia, G., Pantaleo, M., Sciorsci, R.L., 2010. Localization of thyrotropin receptor and thyroglobulin in the bovine corpus luteum. Anim. Reprod. Sci. 118, 1–6. Nixon, D.A., Akasha, M.A., Anderson, R.R., 1988. Free and total thyroid hormones in serum of Holstein cows. J. Dairy Sci. 71 (5), 1152–1160. Rizzo, A., Minoia, G., Trisolini, C., Mutinati, M., Spedicato, M., Manca, R., Sciorsci, R.L., 2009a. Renin and ovarian vascularization in cows with follicular cysts after epidural administration of a GnRH analogue. Anim. Reprod. Sci. 116, 226–232. Rizzo, A., Minoia, G., Trisolini, C., Mutinati, M., Spedicato, M., Jirillo, F., Sciorsci, R.L., 2009b. Reactive Oxygen Species (ROS): involvement in bovine follicular cysts etiopathogenesis. Immunopharmacol. Immunotoxicol., 1–5. Rizzo, A., Campanile, D., Mutinati, M., Minoia, G., Spedicato, M., Sciorsci, R.L., 2011a. Epidural vs intramuscular administration of lecirelin, a GnRH analogue, for the resolution of follicular cysts in dairy cows. Anim. Reprod. Sci. 126, 19–22. Rizzo, A., Spedicato, M., Mutinati, M., Minoia, G., Pantaleo, M., Sciorsci, R.L., 2011b. In vivo and in vitro studies of the role of the adrenergic system and follicular wall contractility in the pathogenesis and resolution of bovine follicular cysts. Theriogenology 76, 1526–1531. Roche, J.R., Friggens, N.C., Kay, J.K., Fisher, M.W., Stafford, K.J., Berry, D.P., 2009. Invited review: body condition score and its association with dairy cow productivity, health, and welfare. J. Dairy Sci. 92, 5769–5801. Roth, Z., Biran, D., Lavon, Y., Dafni, I., Yakobu, S., Braw-Tal, R., 2012. Endocrine milieu and developmental dynamics of ovarian cysts and persistent follicles in postpartum dairy cows. J. Dairy Sci. 95, 1729–1737.
Russell, W., Harrison, R.F., Smith, N., Darzy, K., Shalet, S., Weetman, A.P., Ross, R.J., 2008. Free triiodothyronine has a distinct circadian rhythm that is delayed but parallels thyrotropin levels. Endocr. Res. 93 (6), 2300–2306. Sciorsci, R.L., Rizzo, A., Robbe, D., Minoia, P., 2003. Le cisti follicolari nella bovina. In: Proceedings of the 1st Congress of the Italian Society of Animal Re production (S.I.R.A.), Vico Equense, Napoli, Italy, pp. 98–107. Shu, J., Xing, L., Zhang, L., Fang, S., Huang, H., 2011. Ignored adult primary hypothyroidism presenting chiefly with persistent ovarian cysts: a need for increased awareness. Reprod. Biol. Endocrinol. 9, 119. Silvia, W.J., McGinnis, A., Hatler, T., 2005. A comparison of adrenal gland function in lactating dairy cows with or without ovarian follicular cysts. Reprod. Biol. 5, 19–29. Slebodzinski, A.B., 2005. Ovarian iodide uptake and triiodothyronine generation in follicular fluid. The enigma of thyroid ovary interaction. Domest. Anim. Endocrinol. 29, 97–103. Spicer, L.J., Alonso, J., Chamberlain, C.S., 2001. Effects of thyroid hormones on bovine granulose and thecal cell function in vitro: dependence on insulin and gonadotropins. J. Dairy Sci. 84, 1069–1076. Todoroki, J., Noguchi, J., Kikuchi, K., Ohnuma, K., Ozawa, M., Kaneko, H., 2011. Plasma concentrations of inhibin A in cattle with follicular cysts: relationship with turnover of follicular waves and plasma levels of gonadotropins and steroid hormones. Domest. Anim. Endocrinol. 27, 333–344. Vanholder, T., Opsomer, G., De Kruif, A., 2006. Aetiology and pathogenesis of cystic ovarian follicles in dairy cattle: a review. Reprod. Nutr. Dev. 46 (2), 105–119. Vasseur, C., Rodien, P., Beau, I., Desroches, A., Gerard, C., de Poncheville, L., Chaplot, S., Savagner, F., Croue, A., Mathieu, E., 2003. A chorionic gonadotropin sensitive mutation in the follicle-stimulating hormone receptor as a cause of familial gestational spontaneous ovarian hypersimulation syndrome. N. Engl. J. Med. 349 (8), 753–759. Velázquez, M.M.L., Alfaro, N.S., Salvetti, N.R., Stangaferro, M.L., Rey, F., Panzani, C.G., Ortega, H.H., 2011. Levels of heat shock protein transcripts in normal follicles and ovarian follicular cysts. Reprod. Biol. 11 (3), 276–283.
Contents lists available at SciVerse ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Cystic ovarian follicles and thyroid activity in the dairy cow M. Mutinati, A. Rizzo, R.L. Sciorsci ∗ Department of Emergencies and Organ Transplantation (D.E.T.O.), Faculty of Veterinary Medicine, University of Bari “Aldo Moro”, s.p. per Casamassima, km 3, 70010 Valenzano (Bari), Italy
a r t i c l e
i n f o
Article history: Received 31 October 2012 Received in revised form 20 February 2013 Accepted 28 February 2013 Available online 15 March 2013 Keywords: Dairy cows Cystic ovarian follicles Thyroid hormones
a b s t r a c t Thyroid activity affects the functionality of the reproductive axis and thyroid dysfunction has been associated with ovarian hyperstimulation syndrome and polycystic ovarian syndrome, in human medicine. This study investigates serum17-
1. Introduction Cystic ovarian follicles (COF) represent one of the most common reproductive disorders affecting dairy herd fertility, occurring with an incidence of 6–29%, mainly in the postpartum of dairy cows (Silvia et al., 2005; Vanholder et al., 2006; Grado-Ahuir et al., 2011). According to some authors follicular cysts can be defined as follicle-like structures, with an ovulatory size, persisting for more than 6 days without luteinizing. These structures are likely to undergo spontaneous regression, within the first 50 days postpartum, otherwise they persist or regress being later replaced by another cyst on the same or opposite ovary (Silvia et al., 2005; Rizzo et al., 2009a; Rizzo et al., 2011a).
∗ Corresponding author. Tel.: +39 0805443881; fax: +39 0805443883. E-mail address: [email protected] (R.L. Sciorsci). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.02.024
A large number of factors can influence the development of follicular cysts in dairy cows, in primis an alteration of the hypothalamo-hypophyseal-gonadal axis (Vanholder et al., 2006; Roth et al., 2012), is probably due to an alteration in the feedback mechanism of 17-
M. Mutinati et al. / Animal Reproduction Science 138 (2013) 150–154
expression of heat shock proteins (hsp) in the granulosa and theca cells of the follicular wall (Velázquez et al., 2011) are also contributing factors, to the onset of cystic ovarian follicles, in the dairy cow. In human medicine, thyroid disorders have been associated with ovarian hyperstimulation syndrome (OHSS) (Vasseur et al., 2003; Montanelli et al., 2004; Shu et al., 2011) and with polycystic ovary syndrome (PCOS) (Jung et al., 2011). Both hypothyroidism (Shu et al., 2011) and hyperthyroidism (Jung et al., 2011) have been associated with ovarian cysts in pre-menopausal women. In hypothyroid women developing ovarian cysts, hormone replacement therapy results in a restoration of the euthyroid state as well as a regression of symptoms and ovarian enlargement (Shu et al., 2011). In contrast, Jung et al. (2011) reported that treatment with propylthiouracil is effective in restoring fertility, physiological ovarian appearance and euthyroid function in women with Grave’s hyperthyroidism and PCOS. Therefore this study aimed to investigating steroid, thyrotropic hormone (TSH) and free thyroid hormone (fT3 and fT4) blood concentrations in healthy and infertile dairy cows diagnosed with ovarian follicular cysts.
2. Material and methods In this study all procedures were conducted in accordance with the EU Directive 2010/63/EU. The study was performed between February and July 2012, at a commercial dairy farm in the prefecture of Taranto (Apulian Region, South Italy), consisting of 280 Holstein Friesian cows kept in a semi-intensive regimen and fed pasture, hay, minerals and concentrates; fresh water was available ad libitum. Two clinical examinations were performed 10 days apart using transrectal palpation and ultrasonography (SonoSite, MicroMaxx Bothell, WA, USA, linear probe 5–10 MHz, set at 7.5 MHz). This was used to diagnose cystic ovarian follicles in 30 cows, with a total of 60 cows (including those with CF) chosen and enrolled in the study. The diagnosis of ovarian follicular cysts was performed ultrasonographically, keeping into account the criteria indicated by Silvia et al. (2005), i.e.: mean diameter of at least 17 mm, wall thickness <3 mm, blood progesterone <1 ng/mL, persistence for more than 6 days. The 60 cows chosen, free from common parasites and infective agents, were between the second and third month of lactation (60 ± 10 days post partum) and between the third and the fifth lactation (27 kg/d) and had a mean BCS of 3 (Roche et al., 2009) The choice of cows being in the same, the narrow phase of lactation was chosen as thyroid activity is known change with stage of lactation (Nixon et al., 1988). The cows were divided into two groups of 30 subjects each:
Group CF: cows with ovarian follicular cysts Group H: healthy cows on heat.
151
2.1. Blood samples Blood samples were taken from the coccygeal vein, using vacutainer serum tubes. These were kept on ice and taken to the lab in a mean time of 50 ± 20 min. The blood samples employed for this experimentation were the same used for endocrine routine screening, performed to assess the cystic or cyclic heat condition respectively, and were collected at the following times: • on the day in which ovarian follicular cysts were diagnosed (Group CF); • on the day of heat (Group H). Blood collection was performed at 5:00 pm, in order to avoid the interference of the circadian rhythm on thyroid hormone levels (Russell et al., 2008). Once in the lab, blood samples were centrifuged at 1620 × g for 10 min at +4 ◦ C and sera were stored in eppendorf (1.5 mL) at −20 ◦ C, until analytical determinations. On serum, 17-
2.2. Statistical analysis Statistical analysis was conducted using IBM SPSS Statistics 19 (IBM Corporation Software group, Somers, NY, USA). Student’s T test for independent variables was used for intergroup comparison, whereas Person’s test was employed for correlations among 17-
3. Results The data obtained (Mean ± s.d.) are shown in Figs. 1–5. 17-
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Fig. 1. Serum 17-
Fig. 2. Serum Progesterone concentrations (ng/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.001. Fig. 5. Serum fT4 concentrations (pg/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.01.
(1988) and Nixon et al. (1988), whereas fT4 was found to be a little lower than its physiological levels (Anderson et al., 1988; Nixon et al., 1988). TSH and fT4 serum concentrations were lower in Group FC than in Group H, with TSH and fT4 levels giving rise to statistically significant differences (p < 0.05 and p < 0.01, respectively). Pearson’s test revealed no significant correlations, among the parameters tested, except for TSH and fT3 serum concentrations which were negatively and significantly correlated, in Group H (p < 0.05; R = −0.567). Fig. 3. Serum TSH concentrations (ng/mL) (Mean ± s.d.) in cyclic cows on heat (Group H) and in cows with follicular cysts (Group FC). a,b: p < 0.001.
(Group FC), compared to healthy ones (Group H) (p < 0.01 and p < 0.001, respectively) and confirms what has been previously reported in the literature (Sciorsci et al., 2003). In both groups, serum TSH concentrations were comparable to those found by De Moraes et al. (1998), fT3 concentrations resembled those found by Anderson et al.
4. Discussion and conclusions In the present study, the endocrine milieu in cyclic and cystic dairy cows was investigated. Particular attention was focused on thyroid hormones, since, to the best of our knowledge, only uncertain and old reports dealing with the interaction of thyroid hormones and the establishment of cystic ovarian follicles in the dairy cow, exist (Dzhambazov, 1976).
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In human reproduction, numerous studies have been performed on the interaction among thyroid diseases, infertility and ovarian dysfunctions (Jung et al., 2011; Shu et al., 2011). Furthermore, a strong interaction between thyroid and ovary is inferred from many in vitro researches, both in humans and animals. In fact, the presence of Sodium-Iodide Symporter (NIS) in human and murine ovaries, that of triiodothyronine (T3) and thyroxine (T4) in the ovarian follicular fluid of farm animals, of 5 deiodinase and of thyroid hormone receptors in oocytes, cumuli and porcine and human granulosa cells (Slebodzinski, 2005) have been found. In another study, thyroglobulin (TBG) and TSH receptor were detected in bovine luteal cells by immunohistochemistry (Mutinati et al., 2010). Besides the detection of factors implied in thyroid hormone synthesis and activity, other in vitro studies confirmed a direct role of T3 and T4 in promoting steroidogenesis in human, porcine and bovine follicular and luteal cells (Gregoraszczuk and Galas, 1998; Spicer et al., 2001). In the present workserum fT4 concentrations were statistically lower, in cystic cows, than in cyclic ones, whereas fT3 serum levels were similar in both groups. Free T4 is the major secretory product within the thyrocite, whereas fT3 circulating levels mainly derive from the activity of 5 deiodinase, in peripheral tissues (Kelly, 2000). Given these observations, it is likely that the statistically significant decrease of circulating fT4 found in the cystic cows is due either to a decreased thyroid activity or to an increased activity of peripheral deiodinases. The in vitro studies of Gregoraszczuk and Galas (1998) and Gregoraszczuk (2000) showed that T3 elicits progesterone and androgen secretion in porcine luteal and thecal cells and up-regulation of estrogen receptors in granulose cells. Later, Spicer et al. (2001) demonstrated that T3 is able to enhance aromatase activity in cultured bovine granulose cells, in presence of insulin and FSH. It is well known from literature that cystic cows display higher circulating estradiol and progesterone levels, compared to cyclic ones, according to what occurred in this study (Todoroki et al., 2011; Roth et al., 2012). Given the effects of T3 on steroidogenesis, an involvement of this hormone in the high 17-
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fT3 detected (Nixon et al., 1988), despite the decrease in fT4 serum concentrations found in the cystic group. The reduction in the blood fT4 levels detected in this study may have depended both on a reduction in the thyrotropic activity of TSH and on an increase in peripheral deiodinase activity, which is known to be regulated by TSH (Kelly, 2000). In conclusion, the results of this study confirm that, as in human medicine, also in the bovine species ovarian diseases may be associated with alterations in thyroid activity, but whether thyroid dysfunction is a cause involved in the onset of ovarian cysts, (Jung et al., 2011; Shu et al., 2011) or it is a consequence of the sustained steroid hormone concentration accompanying the cystic condition, is still a matter of debate. Given these results, a thyroid hormone screening could be useful in those cases of CF refractory to common therapies. In this investigation, a slight decrease in fT4 circulating levels, with no effect on fT3 blood concentrations was detected. Should fT3 be lower than its physiologic levels (Anderson et al., 1988; Nixon et al., 1988), a therapeutic approach based on l-thyroxine administration could be attempted, as it routinely occurs in human medicine.
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