Bull fertility is an important aspect because one bull may serve around 20 females under natural conditions or hundreds of thousands under an artificial insemination program (Devkota et al., 2011). Reliable fertility information can be obtained on several bulls by using them to impregnate a large number of cows or by evaluating their semen quality. However, these assessments are time consuming and require the necessary equipment and skills. The inadequacies of these techniques has compelled researchers to identify simple and reliable parameters to predict sperm output and fertility. Testicular weight is one among such parameters that provides an accurate estimate of the amount of sperm-producing parenchyma in the testis (Amann and Almquist, 1962; Coulter and Foote, 1977). In live bulls, testicular weight could be derived by indirect measurements such as testicular length and width and scrotal circumference (Bailey et al., 1998).

Thyroid hormones are critical regulators of growth, development, and metabolism in virtually all tissues, and altered thyroid status affects many organs and systems. For a long period of time, testis was regarded as an organ unresponsive to thyroid hormones (Wagner et al., 2008). However, recent studies have demonstrated its role in testicular development and function. The active thyroid hormone receptor isoforms (TRα1 and TRβ1) are present in Sertoli, Leydig, peritubular, and germ cells of testis (Buzzard, 2000; Canale et al., 2001; Rao et al., 2003). Thyroid hormone inhibits the proliferation and stimulates the differentiation of immature Sertoli cells during the post-natal period (Matta et al., 2002; Jansen et al., 2007). It also inhibits the proliferation of mesenchymal Leydig cell precursors and promotes the formation of adult Leydig cells (Teerds et al., 1998; Ariyaratne et al., 2000). Triiodothyronine increases the basal as well as LH (luteinizing hormone)-stimulated testosterone production by upregulating the enzymes involved in the conversion of cholesterol to testosterone (Maran et al., 2000; Manna et al., 2001).

Although the literature indicates the involvement of thyroid hormones in the development of testis and the production of testosterone, very few in vivo studies have been conducted to determine their association. With this perspective, the present study was designed to investigate the developmental changes in testicular parameter and its association with thyroid hormones and testosterone in male Murrah buffaloes.

The plasma concentrations of T4 and T3 and their trend lines in relation to age are represented in Figs. 1 and 2, respectively. The plasma T3 and T4 levels ranged between 0.38 and 2.04 and 12.9 and 41.8 ng mL-1, respectively. The regression equation between T4 concentrations and age was Y=12.6+0.02x-2.67×10-6x2-2.21×10-10x3 (r= 0.61; R2= 0.37). The regression equation between T3 concentrations and age was Y=0.95+0.001x-3.67×10-7x2+6.40×10-11x3 (r= 0.2; R2= 0.04). The results indicated a moderate relation between T4 concentrations and age, whereas a poor relation between T3 concentrations and age was observed. We did not observe a definite trend line of increase or decrease in plasma T3 levels with age, which was in agreement with a previous study conducted on Murrah buffaloes (Pandita et al., 2016).

We observed a significant (p< 0.001) increase in plasma testosterone with age and its levels ranged between 0.05 and 1.48 ng mL-1 (Fig. 3). The regression equation between testosterone concentrations and age was Y=-0.10+0.002x-1.06×10-6x2+1.85×10-10x3 (r= 0.58, R2= 0.34). The age-related changes in testosterone concentrations, range of testosterone, and the correlation coefficient value observed in the present study were in accordance with a similar study carried out on male Murrah buffaloes (Gulia et al., 2010). A linear relation in plasma testosterone with age was observed among the animals aged between 192 and 708 days. Similar changes in plasma testosterone were also noticed in investigations conducted on Egyptian buffalo bulls (Hemeida et al., 1985), Angus bull calves (Moura et al., 2001), Angus and Angus × Charolais bull calves (Brito et al., 2007), and Japanese Black bull calves (Kawate et al., 2011).

The average TW in relation to age ranged between 9.86 and 403 g (Fig. 4). The regression equation between average TW and age was Y=1.48×x0.005 (r= 0.90; R2= 0.79). The dispersion of testicular length, width, and circumference in relation to age and their regression equations are depicted in Fig. 5. The average testicular length (ATL), paired testicular width (PTW), and SC in relation to age ranged between 4.50 and 16.0, 4.00 and 14.0, and 11.0 and 36.0 cm, respectively. The correlation coefficients of ATL, PTW, and SC in relation to age were 0.88, 0.91, and 0.90, respectively. A similar positive correlation of testicular measurements and indices with age was also observed in Holstein (Coulter et al., 1975; Coulter and Foote, 1976), Angus (Coulter et al., 1975; Coulter and Keller, 1982), Hereford (Coulter and Keller, 1982; Menegassi et al., 2011), Charolias (Menegassi et al., 2011), Sahiwal (Ahmad et al., 2011), and Murrah buffalo (da Luz et al., 2013) males. The age-related distribution of testicular parameters (Fig. 5) observed in the present study agrees with the findings of a previous study (Coulter et al., 1975), which inferred that testicular size increases rapidly in young bulls and more gradually in mature bulls.

The associations between testicular parameters, plasma testosterone, T3, and T4 are given in Table 1. We observed significant (p< 0.01) correlations between plasma T4 and testicular parameters. A significant correlation (r= 0.31; p< 0.01) between plasma T4 and testosterone levels was also observed. However, the correlations between plasma T3 and testicular parameters and plasma T3 and testosterone were non-significant. Although T3 is the bioactive form that exerts the developmental (Ariyaratne et al., 2000; Jansen et al., 2007) and steroidogenic effects (Maran et al., 2000; Manna et al., 2001) on testicular tissue, T4 also participates directly in testicular development by promoting amino acid accumulation in Sertoli cells (Menegaz et al., 2006). Moreover, most of the T4 is converted to T3 in the testicular tissue. The deiodinases (D1 and D2) involved in the conversion of T4 to T3 are expressed in testis from weanling to adult life (Bates et al., 1999).

From the present study, we conclude that plasma T4 levels are positively correlated with testicular parameters and plasma testosterone, indicating the developmental and steroidogenic effects of thyroid hormones on the testis.