Association between blood and seminal plasma testosterone and aldosterone and their consequences on semen analysis and fertility status in dromedary camel

doi:10.21203/rs.3.rs-1413974/v1

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Association between blood and seminal plasma testosterone and aldosterone and their consequences on semen analysis and fertility status in dromedary camel

https://doi.org/10.21203/rs.3.rs-1413974/v1

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This study aimed to investigate the testosterone and aldosterone profiles in the blood and seminal plasma of male dromedary camels and their relationship with semen parameters. Sixty camels were divided into two groups; control (n = 20) and infertile (n = 40). Blood and seminal plasma were obtained from all camels for hormones analyzed. The results indicated significant higher (p < 0.05) blood testosterone (3.05 ± 0.12 vs. 5.17 ± 0.24 ng/mL), and seminal plasma testosterone concentrations (1.29 ± 0.22 vs. 2.78 ± 0.29 ng/mL) in infertile above control dromedaries, respectively. In the control group, there was a positive correlation (p < 0.05, r = 0.36) between blood and seminal plasma aldosterone. In infertile males, there found a positive correlation (p < 0.01, r = 0.45) between blood testosterone and aldosterone. Also, there existed a negative correlation (p < 0.05, r = − 0.26) between blood aldosterone and seminal plasma testosterone concentrations. In control males, there was a negative correlation (p < 0.05, r = − 0.42) between blood aldosterone and sperm motility percentage. Besides, seminal plasma aldosterone was positively correlated (p < 0.05, r = 0.26) with sperm abnormality ratio in the infertile animals. In conclusion, infertile male dromedaries were characterized by elevated testosterone levels in the blood and seminal plasma. In addition, there found a positive relationship between blood testosterone and aldosterone, as well as a negative association between blood aldosterone and seminal plasma testosterone. Thus, these parameters could be considered as bio-indicators for spermatogenic impairment and disruption of seminal plasma bio-products, resulting in poor-quality semen ejaculates.

Dromedaries

Semen quality

Aldosterone

Testosterone

Fertility rate

In the harsh environmental conditions in the desert, semi-desert, and tropical regions, dromedary camels (Camelus dromedarius) can survive and provide herders with valuable products such as milk, meat, and hair (El-Khaldi and Homeida, 2020). The camels are capable of surviving water deprivation in an extraordinary way. A large part of this can be attributed to the fact that it can alter the production of hormones that affect sodium and water homeostasis (Ali et al. 2012). In most circumstances, this system serves to retain water and/or sodium and maintain circulatory and renal status through arginine vasopressin, aldosterone, and renin-angiotensin (Ali et al. 2012). Also, due to their low evaporative cooling, low urinary excretion, and their ability to recoup water from food residues, camels are capable of reducing water turnover (Yagil, 1993). Furthermore, the kidneys of camels play an important role in water retention by producing highly concentrated urine, which may predispose camels to various kidney diseases (Tharwat, 2020). In addition, dehydration has been found to cause significant increases in serum sodium, creatinine, urea, and arginine vasopressin levels, as well as decreases in aldosterone levels, which block angiotensin II (Ang II) type 1 receptors and enhance weight loss in dromedary camels (Ali et al. 2012). Thus, the renin-angiotensin system (RAS) is a peptide system primarily responsible for the maintenance of blood pressure and electrolyte and fluid homeostasis (Paul et al. 2006). The RAS system is considered a circulating hormonal system that exerts its functions through angiotensin II (Ang II) and aldosterone (Paul et al. 2006).

Dromedaries are considered seasonal breeders and their breeding season is limited to the winter of the year (Skidmore, 2005). Although individuality could be observed morphologically, behaviorally, and in the hormonal changes throughout the breeding season (Farh et al. 2018). Short camel breeding season, low libido, and aggression can still lead to poor reproductive performance and economic loss (Farh et al. 2018). Camel reproductive efficiency is also reported to be less than that of other domesticated animals (Kaufmann, 2005; Skidmore, 2005). Furthermore, impotence is a common infertility problem in male dromedary camels (Ali et al. 2014). Dromedary camels diagnosed with impotence were characterized by failure in spermatogenesis due to oligospermia and azoospermia (Ali et al. 2018). In addition, impotent camels had higher blood levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone, which inversely correlated with reduced testicular size and sperm count (Ali et al. 2018). However, the cause of male dromedary infertility problems remains a mystery.

Multiple RAS family members are expressed on different male reproductive tissues, such as testis, sperm, and semen (Paul et al. 2006). They regulate male fertility, synergistically with and/or independent of systemic RAS (Paul et al. 2006). In addition, some of the functions recognized by this local RAS are maintenance of seminal plasma electrolytes, regulation of steroidogenesis, spermatogenesis, and sperm function (Gianzo and Subirá, 2020). However, the exact mechanisms by which they are located at these locations have not been resolved. Furthermore, to our knowledge, there is no information on the relationship between the RAS system and/or the regulation of fertility in dromedary male camels. Besides, even conventional methods of measuring semen quality can identify some infertile dromedary camels. However, there is considerable variations relative to in vivo fertility among camels considered fertile. Consequently, more diagnostic tests are still needed to determine the cause of infertility in dromedary camels. Therefore, this study aimed to explore the changes of testosterone together with aldosterone in the blood and seminal plasma of male dromedary camels and their relationship with semen parameters as well as fertility status. In this study, we hypothesized that dehydrated conditions in the desert could modify the production of aldosterone through the RAS system, which plays a crucial role in maintaining blood pressure, electrolyte, and fluid homeostasis, and may even contribute to the regulation of fertility and sperm function in male dromedary camels.

Animals and experimental design

The present study examined sixty male dromedary camels (Camelus dromedarius) in private camel farms in the Qassim region, central Saudi Arabia. Animals aged 6 to 13 years with bodies weighed between 460 and 618 kg, with a body condition score of 3–4.5 on a scale of 5. All animals were kept under nomadic conditions in an open yard during the breeding season (Nov-Feb). All studied camels were maintained under standard feeding conditions (3 kg mixture of barley and wheat bran per day, Rhodes grass and water were provided ad libitum). Using the breeding history records, the camels were divided into two groups: Control (served as sires active for breeding females, n = 20) and infertile (unable to achieve conception, even after several attempts during the breeding seasons, n = 40). Camel fertility was evaluated using the camel fertility index (the number of females mated with this camel divided by the number of that conceived). Following the guidelines of the Animal Welfare Committee, the experimental procedures were approved by the University Ethics Monitoring Committee.

Blood sampling, Semen collection and evaluation

Blood samples were collected from the jugular vein in a heparinized tube from all camels, centrifuged at 4000 ×g for 10 min, and plasma was harvested, aliquot, and stored at -20°C until analyses were performed.

Semen was collected from all animals by an electro-ejaculator (ElectroJac 6; Neogen, Lexington, KY, USA) as described previously (Ali et al. 2014). The semen ejaculate was tested by using ISAS program (CASA; Proiser & Ziotech, Madrid, Spain) for usual semen quality parameters such as volume (mL), viscosity, sperm concentration, percent sperm abnormalities, and percent progressively motile sperm as explained earlier (Waheed et al. 2015). The seminal plasma was separated by centrifugation at 4000 ×g for 10 min, aliquot and stored at -20°C until analyzed.

Hormone determinations

Testosterone levels in the blood and seminal plasma were measured by a commercial ELISA kit (Absorbance Microplate Reader ELx 800 BioTek, USA; Microplate Strip Washer ELx 50 BioTek, USA). The coefficients of variation for intra-assay were 5.9%, and the sensitivity of the assay was 0.20 ng/mL. Similarly, blood and seminal plasma aldosterone levels were measured using a commercial ELISA kit (Diagnostics Systems Laboratories Inc., TX, USA), with an intra-assay coefficient of variation of 4.5% and sensitivity of 10 pg/mL.

Statistical analysis

All data are shown as means ± SEM. These data were analyzed by Student’s t-test (t) for comparisons between the two tested groups, and correlations coefficients (r²) were tested using the SPSS program, version 24.0 (SPSS, 2016). The differences were considered to be statistically significant at p < 0.05.

Blood testosterone level was lower (p < 0.05) in control “fertile” than that in infertile camels (3.05 ± 0.12 vs. 5.17 ± 0.24 ng/mL, Fig. 1A). Similarly, lower (p < 0.05) testosterone levels were found in the seminal plasma of control (1.29 ± 0.22 ng/mL) compared to infertile (2.78 ± 0.29 ng/mL, Fig. 1B) males. In contrast, there were no differences (p > 0.05) in aldosterone levels in the blood or seminal plasma among the two groups (Fig. 2A, B). Furthermore, in case of fertile control camels, a positive correlation (p < 0.05, r = 0.36) was recognized between blood aldosterone and seminal plasma aldosterone levels. However, in the infertile males, a positive correlation (p < 0.01, r = 0.45) was obtained between blood testosterone and blood aldosterone levels. Additionally, a negative correlation (p < 0.05, r = − 0.26) was demonstrated between blood aldosterone and seminal plasma testosterone levels in infertile camels.

The percentage of sperm motility and viability were lower (p < 0.05) in the infertile camels compared to control camels (Table 1). Moreover, abnormal sperm value was higher (p < 0.05) in the infertile than in the control fertile males (28.26 ± 3.84% vs. 8.20 ± 1.20%). Besides, sperm cell concentration was lower (p < 0.05) in the infertile compared to control males (Table 1). However, mean ejaculate volume did not differ (p > 0.05) between both groups. Furthermore, in control fertile camels, a negative correlation (p < 0.05, r = − 0.42) was shown between blood aldosterone and sperm motility percentage. Contrariwise, in the infertile males, a positive correlation (p < 0.05, r = 0.26) was observed between seminal plasma aldosterone and sperm abnormality ratio.

Table 1

Semen characteristics (mean ± SE) of control and infertile dromedary male camels
Item	Control	Infertile
Volume (ml)	5.63 ± 0.27	4.90 ± 0.85
Sperm motility (%)	64.60 ± 1.41^a	27.64 ± 2.91^b
Sperm viability (%)	71.60 ± 2.62^a	34.54 ± 3.98^b
Sperm abnormality (%)	8.20 ± 1.20^a	28.26 ± 3.84^b
Sperm count mm³ (× 10³)	624.60 ± 12.22^a	454.50 ± 12.67^b
^{a, b} Means within the same row with different superscripts are significantly different at P < 0.05.

In the present study, the increased testosterone levels in both blood and seminal plasma of the infertile compared with control fertile male camels, suggests that infertile camels could have primary defects in the spermatogenesis process (Babu et al. 2004; Ali et al. 2018). Other possible explanation for this elevated testosterone levels could be due to the increased density of the interstitial cells on the expense of the seminiferous tubules (Ali et al. 2018). Moreover, changes in the structure and function of Leydig cells accompanying seminiferous tubule damage could explain the rise in blood plasma testosterone concentrations of infertile camels (Tibary, 2004; Ali et al. 2014) which is reflected in the seminal plasma. The high blood testosterone levels may also be related to a rise in LH levels that causes androgen resistance in the infertile dromedary male camels (Fraietta et al. 2013). Contradictory to the previous findings, Waheed et al. (2015) reported a higher testosterone concentration in the serum blood of fertile compared to infertile male dromedary camels. Previous studies suggested that male infertility can be caused by a variety of conditions; such as gonadotropin deficiency (Babu et al. 2004), primary testicular failure (Ali et al. 2014), spermatogenic failure (Bhasin et al. 2007), or androgen resistance (Fraietta et al. 2013). Furthermore, in the present study, significant rises in seminal plasma testosterone levels of infertile camels support the suggestion that those animals could be showing a status of spermatogenic failure, and/or androgen resistance conditions. Thus, testosterone in both seminal plasma and blood of dromedary camels could be used as fertility-associated biomarkers (Waheed et al. 2015).

In the current study, no difference was observed in aldosterone levels in the blood or seminal plasma among both groups. However, aldosterone levels are slightly altered by dehydration in dromedary camels (Ali et al. 2012). Also, aldosterone is the chief regulator of sodium, potassium, and chloride metabolism, thus controlling the body's water and electrolyte balances (Yang and Ma, 2009). Accordingly, the detected aldosterone level in the present study suggested that both fertile and infertile camel males had similar environmental conditions regarding dehydration and water balances. Also, the equal response in circulating aldosterone levels in both groups can be explained by the equal amount of sodium in the serum and presumed osmolality in plasma, though unmeasured. In addition, the current results confirmed a positive association between blood plasma testosterone and aldosterone in infertile camels. Inversely, in vivo and in vitro experiments in male rats (Kau et al. 1999), suggested that testosterone inhibits the basal and Ang II- and corticotropin (ACTH)-stimulated release of aldosterone. This mechanism occurs via inhibition of aldosterone synthase activity and cytochrome P-450 side-chain cleavage (P450scc) activity, and ACTH-stimulated c-AMP accumulation in the zona glomerulosa cells. Also, endogenous androgens (testosterone) exert anti-hypertensive effects that appear to involve non-genomic and possibly genomic mechanism(s), resulting in reductions in RAS expression in the kidney and enhanced systemic vasodilation in male rats (Hanson et al. 2020). Instead, the present study indicated a negative association between blood aldosterone and seminal plasma testosterone in infertile camels.

According to the current findings, there was a significant difference in the motility, viability, concentration, and abnormalities of sperm and the fertility status between the control and infertile male camels. Similar data were reported previously in dromedary camels (Mostafa et al. 2014, Waheed et al. 2015; Waheed et al. 2018). Several factors can contribute to poor semen quality, including impaired spermatogenesis, endocrine malfunctions, alteration of the microenvironment of seminiferous tubules, and accessory gland infections (Robaire and Hamzeh, 2011). Also, it has been reported that testicular hypoplasia is an inherited trait in the dromedary camels, characterized by the absence of or the atretic seminiferous tubules of the testicular parenchyma resulting in spermatogenic failures (Tibary, 2004; Ali et al. 2014). Furthermore, decreased sperm motility and concentration, as well as increased abnormalities, were associated with higher iron (Fe), zinc (Zn), and copper (Cu) concentrations in serum and seminal plasma of infertile camels (Waheed et al. 2018). Besides, higher levels of arsenic (As), cadmium (Cd), and lead (Pb) in seminal plasma and blood were associated with lower levels of semen quality variables and infertility in dromedary camels (Meligy et al. 2019). Likewise, the present study showed a negative association between blood plasma aldosterone and sperm motility in control camels. Also, a positive relationship was indicated between seminal plasma aldosterone and sperm abnormality in the infertile camels. A possible link between aldosterone and semen quality is the local RAS regulator of seminal plasma electrolytes, steroidogenesis, and spermatogenesis, as well as sperm function (Gianzo and Subirá, 2020). Also, the RAS acts locally through different paracrine and autocrine mechanisms (Gianzo and Subirá, 2020). Yet, the definitive explanation for aldosterone's influence on the semen quality of the dromedary camel still needs to be resolved.

Infertility cases in male dromedary camels are characterized by elevated testosterone levels in the blood and seminal plasma. Apparently, the reverse relationship between blood aldosterone and seminal plasma testosterone could be used as a biomarker for early diagnosis of subfertility statuses in camel males. Besides, the positive relationship between seminal plasma aldosterone and sperm abnormality would also be considered in this matter. Subsequently, infertile male dromedary camels could be showing a status of spermatogenic failure, disruption of seminal plasma electrolytes, and/or androgen resistance, leading to producing poor quality semen and low fertilizing capacity.

Author contribution: Zeitoun M.M. and Mansour M.M. conceived and designed research. Zeitoun M.M. conducted experiments. Mansour M.M analyzed data. Zeitoun M.M. and Mansour M.M. wrote the manuscript. All authors read and approved the manuscript.

Ethics approval: Following the guidelines of the Animal Welfare Committee, the experimental procedures were approved by the Qassim University Ethics Committee, Saudi Arabia Kingdom.

Funding: “The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.”

Conflict of interest: The authors declare no competing interests.

Availability of data and materials: The authors ensure that all data and materials support the findings and comply with field standards. The farmer was informed of the study, and he gave his consent to data collection from his farm and animals.

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You are reading this latest preprint version

Association between blood and seminal plasma testosterone and aldosterone and their consequences on semen analysis and fertility status in dromedary camel

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