Spectral Doppler ultrasound analysis of the testicular artery showed a tendency towards a lower EDV in low RFI animals when compared to high RFI animals. The PSV and EDV represent the velocity at which blood flows through the analyzed blood vessel and reaches the tissue. According to Ortiz-Rodriguez et al. (2017), the higher the velocity at which blood passes through the testicular artery, the better the blood perfusion in the testis. Studies have reported a relationship between blood flow velocity and male reproductive capacity in different animal species (Kutzler et al., 2011; Ortiz-Rodriguez et al., 2017; Gacem et al., 2020; Lemos et al., 2020).
In the present study, higher RI and PI were observed in low RFI animals compared to high RFI animals. According to Pozor and McDonnel (2004), PI and RI are more sensitive markers of blood flow than EDV and PSV since these indexes provide not only information about velocity but also about vascular impedance. The PI and RI represent the difficulty of blood to flow through the vessel; the higher these indexes in the testicular artery, the lower the testicular tissue perfusion and consequently the supply of oxygen and nutrients to the testes (Strina et al., 2016; Fávaro et al., 2020). Parenchymal organs require continuous blood flow and the arteries that supply these structures typically have low resistance (Carvalho et al., 2008).
Analysis of pixel intensity of the testicular parenchyma revealed no differences between high and low RFI animals. This result corroborates the findings of Kowalski et al. (2017) who did not observe a difference between RFI classes in young developing Purunã bulls. On the other hand, Fontoura et al. (2016) and Bourgon et al. (2018) found higher maximum pixel intensities in animals with lower feed efficiency (high RFI). This difference might be related to the age of the animals in the cited studies since pixel intensity of the testicular parenchyma is higher before and during puberty (Brito et al., 2004; Rodrigues et al., 2020). The hypothesis to explain this difference is that spermatogenesis starts at a certain stage of development of the testicular parenchyma during puberty (Kastelic and Brito, 2012). Furthermore, the breed may also be a determinant factor, as reported by Rodrigues et al. (2020) who observed differences in testicular pixel intensity between zebu (Nellore) and taurine (Caracu) animals.
Observing the ultrasound results, the tendency towards a difference (P=0.061) between RFI classes might be related to differences in the number or diameter of the seminiferous tubules, which could affect the heterogeneity of the testicular parenchyma (Brito et al., 2012). The testicular tissue is homogenous and moderately echogenic. This state can change during puberty or in the presence of testicular pathology that can alter homogeneity and increase the pixel intensity as a result of fibrotic processes (Kastelic and Brito, 2012). Despite these differences in the ultrasound parameters of the testicular artery and vascular parameters of the pampiniform plexus between low and high RFI animals, they were not sufficient to cause differences in SC or in the quality of sperm motility in these animals.
The mean SC did not differ between low and high RFI animals. Similar results have been reported in previous studies comparing bulls with distinct RFI values (Hafla et al., 2013; Wang et al., 2012; Fontoura et al., 2016; Kowalski et al., 2017). On the other hand, Awda et al. (2013) and Bourgon et al. (2018) observed a greater SC in high RFI animals but the difference decreased when the animals received better-quality diet, suggesting that this difference in reproductive parameters between low and high RFI animals is due to the energy distribution for maintenance and production and reproductive traits. The authors suggested that, in animals with low RFI, reproductive parameters may have a lower priority, a fact delaying sexual maturity.
Although several studies have associated vascular parameters with semen quality (Gloria et al., 2018; Hedia et al., 2019; Gacem et al., 2020), the higher EDV in least efficient animals and the higher RI and PI in most efficient animals observed in the present study were not sufficient to cause alterations in the seminal parameters studied. Evaluation of the parameters of fresh and thawed semen and after TRT showed that feed efficiency did not affect sperm kinetics since no differences in CASA parameters were detected between low and high RFI animals. The results of the present study corroborate other studies that evaluated sperm motility or progressive motility in low and high RFI animals (Hafla et al., 2013; Awda et al., 2013; Fontoura et al., 2016; Bourgon et al., 2018). However, some authors changed the division of low and high RFI classes and reported different results. Including body composition traits in the equation for calculating RFI, Fontoura et al. (2016) found higher total and progressive sperm motility in least efficient (high RFI) animals when compared to most efficient (low RFI) animals, which was not observed in the present study. On the other hand, Wang et al. (2012) observed lower sperm motility in low RFI animals. However, this difference was not sufficient to reduce the fertility of breeding animals, with the most efficient animals having a larger number of offspring. It should also be noted that the mean values reported by the cited authors are considered excellent for andrological examination (Kennedy et al., 2002; Penny, 2010; CBRA, 2013).
When the sperm morphology of fresh semen was analyzed, we found only differences in the percentage of minor defects, with most efficient (low RFI) animals exhibiting a smaller number of defects than least efficient (high RFI) animals. This result contradicts most of the studies that compared sperm morphology between low and high RFI bulls and did not observe any difference (Wang et al., 2012; Bruinjé et al., 2019) or observed higher percentages of sperm pathologies in low RFI animals (Hafla et al., 2013; Fontoura et al., 2016; Bourgon et al., 2018). Despite the significant difference in sperm morphology observed here between animals with distinct RFI, the mean values are within the range recommended by different andrology handbooks (Kennedy et al., 2002; Penny, 2010; CBRA, 2013; Chenoweth; McPherson, 2016).
The percentages of spermatozoa with a stable plasma membrane, with intact plasma and acrosome membranes, and with high mitochondrial potential were similar between low and high RFI animals. These results corroborate some of the findings reported by Bruinjé et al. (2019); however, these authors reported a higher percentage of mitochondrial respiration activity in spermatozoa from low RFI animals, while the proportion of cells with low mitochondrial potential was higher in these animals. Other studies observed higher rates of mitochondrial activity in liver tissue (Lancaster et al., 2014), in longissimus dorsi muscle (Kolath et al., 2006), and in lymphocytes (Ramos; Kerley, 2013) of most efficient animals (low RFI).
In the present study, although there was no difference in the proportion of cellular respiration of sperm between the two RFI classes, most efficient animals (low RFI) exhibited a better quality of cellular respiration of stable cells in cryopreserved semen. These results may explain why the lower blood flow observed in the testicular artery of low RFI animals was not sufficient to change the sperm kinetics of fresh or thawed semen. One hypothesis would be that sperm cells of low RFI bulls are more efficient in energy production, requiring less blood supply, or that they are adapted to a lower nutritional demand since mitochondria can be partially influenced by the surrounding environment, particularly by other organelles (Keil et al., 2011).
The results suggest that RFI does not influence sperm kinetics nor the sensitivity of sperm to cryopreservation; however, feed efficiency influences blood flow in the vascular cone, increasing the difficulty of blood to pass through the testicular arteries and to reach the testes in most efficient animals. However, cellular metabolism may have compensated for the lower availability of nutrients for sperm cells.
In conclusion, low RFI bulls have lower blood flow in the pampiniform plexus, resulting in greater heterogeneity of the testicular parenchyma evaluated by B-mode and Doppler ultrasound. On the other hand, the reduced blood flow in the pampiniform plexus of low RFI bulls was not sufficient to change sperm kinetics, indicating that the RFI class does not affect the quality of fresh semen, thawed semen, or semen after rapid TRT.