This investigation was designed to evaluate the effect of the hot summer season on the fertility of Arabian stallion of different ages. Ejaculates and blood samples were collected at weekly interval from young (n=6, 5-6 years), middle (n=6, 11-12 years) and aged (n=6, 15-20 years) stallion groups. The gel-free portion of the ejaculate was evaluated for volume, spermatozoa motility, concentration and abnormalities. Plasma samples were analyzed for testosterone, lipids (cholesterol, triglyceride), total protein, oxidative stress biomarkers (catalase, superoxide dismutase, glutathione reduced, malondialdehyde, and total antioxidant capacity), micro-elements (copper, zinc) and macro-elements (calcium, phosphorus, sodium, potassium).Testosterone hormone (p=0.07), semen pH (p<0.005), normality (p<0.001), individual motility (p=0.08), tail abnormalities (p<0.001), total sperm count per ejaculate, total antioxidant capacity (p<0.05), phosphorus (p=0.06) and sodium (p<0.05) noticeably affected by stallions age during the hot summer season. Testosterone level was higher in middle age stallions than young age group. The youngest age group had low semen pH, tail abnormalities, total sperm count per ejaculate and phosphorus levels, but had higher semen volume and spermatozoa individual motility and sodium levels. Stallion of middle and old ages had a significant higher cholesterol level than young aged group. Total anti-oxidant capacity was higher in the old-age group than young and middle aged groups. These results suggest that the stallion fertility is extremely altered by the age during hot summer breeding season, through the metabolic (cholesterol level) status, oxidative stress resistance (total anti-oxidant capacity) and macro-minerals (phosphorus and sodium). It is recommended to implicate middle age stallions in the natural breeding purposes due to the lowest fluctuation of its semen picture, high testosterone-dependent sexual behavior and oxidative stress resistance.
Keywords: Arabian stallion, Minerals, Oxidative stress markers, Spermiogram, summer season, Testosterone
1. Introduction
Horses, long-day seasonal breeders, exhibit annual cycles of breeding activity, seasonal changes in testicular size, sperm production (Clay et al., 1987), seminal pH and sex drive depicted by the reaction time and mounts per ejaculate (Abou-Ahmed et al. 1993).
Many physiological and ambiental factors affect some features of stallion semen, among which the photoperiod is one. The volume of semen, sperm count and spermatozoa motility of fresh semen has been shown to be higher in summer than in winter (Janett et al. 2003). Stallion semen characteristics, except of color, had significantly varied with age and the poorest semen was found in stallions under 3 years and over 11 years of age (Dowsett and Knott, 1996).
As endotherms, mammals typically function at high core body temperatures that range from approximately 35°C to 39°C (Prosser and Heath, 1991). Body temperature is closely regulated by matching heat production with heat loss to the environment via conduction, convection, radiation and evaporation. The set-point temperature for regulation of body temperature is not fixed, but can vary diurnally or, in some animals, in response to changes in environmental temperature (Heldmaier et al., 2004). A stallion’s testicles are close to the abdominal wall, and the internal temperature of the scrotum stays at several degrees cooler than the core body temperature, a necessity for normal spermatogenesis (Setchell et al., 1994).
The summer months bring many enjoyable horse-related activities, but they also bring heat and humidity. Heat stress has large effects on most aspects of reproductive function in mammals. These include disruptions in spermatogenesis and oocyte development, oocyte maturation, early embryonic development, fetal and placental growth and lactation (Hansen, 2009). Exposure of the testes to elevated temperatures for prolonged periods may cause certain stages of developing sperm to be affected and die, resulting in a decrease in sperm numbers in the ejaculate or an increase in the percentage of morphologically abnormal sperm. Researchers have evaluated how the scrotum and its structures can relieve elevated temperatures brought about by heat stress (from environmental stress, exercise or sickness) (Mawyer et al., 2012; Rosenberg et al., 2013).
Many horse owners have questioned how summer heat stress may affect their horse’s reproductive performance. Information about the effect of summer season condition on reproductive characteristics of stallions of different age is scarcely available. Therefore, the aim of the present study was to evaluate the changes in semen characteristics, plasma concentrations of testosterone hormone, lipid and protein profile, oxidative stress markers and minerals’levels in Arabian stallion of different ages during hot summer breeding season.
2. Material and methods
2.1. Semen collection and evaluation
The present study was conducted at Al Zahraa Farm, Ain Shams, Cairo (30°06′ N and 31°25′ E; Latitude30.05), Egypt during the period August-September, 2015.
All experimental procedures were conducted according to the guidelines of the Ethics for humane treatment of animal use in research and complies with the relevant legislation of Faculty of Veterinary Medicine, Benha University, Egypt.
Arabian stallions (n=18) were categorized according to their age into 3 groups, each of six stallions; group I (5-6 years), group II (11-12 years) and group III (15-20 years). Three ejaculates were collected from each stallion at weekly interval, early in the morning, with the use of behaviorally estrus mares as mount animals.
Semen was collected using a lightly lubricated and pre-warmed (45-48°C) Missouri model artificial vagina with an inline filter to separate the gel fraction. Semen samples were immediately transferred to a well equipped laboratory prepared so that all equipments used in post-collection semen handling at 35-37 °C. The gel-free portion of the ejaculate was evaluated visually for volume, and microscopically for progressive motility, concentration (Kenney et al., 1983) and the morphological abnormalities using eosin-nigrosin stain (Dowsett et al., 1984) by conventional methods. Total sperm count per ejaculate (semen volume times concentration) was calculated from the volume and sperm concentration. The pH was determined with waterproof pocket pH tester (HI98107, Hanna, USA)
2.2. Blood sampling and analytical methods
Blood samples were collected from the external jugular vein into EDTA contained vacuum tubes. Immediately after collection, the blood samples were centrifuged at 1500 rpm for 15 min, and the harvested plasma was kept at −20°C until being analyzed.
2.2.1. Hormonal analysis
Testosterone (Cat. No. BC-1115, BioCheck Inc.,CA, USA) level was estimated in plasma with the use of the enzyme immunoassay test kit according to the manufacturer’s instructions.
2.2.2. Biochemical analysis
2.2.2.1. Lipid and protein profile:
Plasma cholesterol (Cat. No. CF03000050, Centronic GmbH, Germany,) and triglyceride (Stanbio Triglyceride LiquiColor® Test, Cat. No SB2200225, AKF Diagnostic Co., USA) were determined spectrophotometrically (UV-120-12, Shimadzu Corp., Kyoto, Japan) after enzymatic hydrolysis according to Richmond (1973) and Whalefeld (1974), respectively. Total protein (Cat. No. PF04000050, Centronic GmbH, Germany) was measured colorimetrically using Biuret reaction according to Layne (1957).
2.2.2.2. Oxidative stress markers
Kits for assessing the activity of catalase (CA 2517) and superoxide dismutase (SD 2521) enzymes as well as glutathione reduced (GR 25 11), lipid peroxidation; malondialdehyde (MD 2529) and total antioxidant capacity (TA 2513) were purchased from Biodiagnostic and Research (Egypt). Catalase (Aebi, 1984) and superoxide dismutase (Nishikimi et al., 1972), glutathione reduced (Beutler et al., 1963), malondialdehyde (Satoh, 1978) and total antioxidant capacity (Koracevic et al., 2001) were evaluated spectrophotometrically as described formerly at 510 nm, 560 nm, 405 nm, 534 nm and 505 nmagainst control, respectively.
2.2.2.3. Micro-and macro-elements
Plasma copper (µmol/L), zinc (µmol/L), calcium (mmol/L), phosphorus (mmol/L) sodium (mmol/L) and potassium (mmol/L) was measured by atomic absorption spectrophotometry (Perkin-Elmer 2380, USA) according to Fernandez and Kahn (1971). Instrument warm-up and optimization were carried out as detailed in the operating manual. The selected wavelength for each element detection was 324.80, 213.90, 422.70, 213.55, 589.00 and 766.50 nm, respectively.
2.3. Statistical analysis
The data were analyzed and presented as mean ± SEM with one-way analysis of variance (ANOVA) using SPSS (Ver. 16). Multiple comparisons of the means were done with least significant difference test. P value was set at <0.05 to define statistical significance
3. Results
3.1. Changes in hormonal levels and semen characteristics in stallions of different ages during summer season
Data regarding hormonal levels and semen characteristics in stallions during summer season is presented in table 1.Assessment of testosterone hormone declared that the stallion age tended (p=0.07) to significantly impact the circulating hormonal level, where the middle age group had a significant (p<0.05) higher level than that of the younger age group (0.50±0.08 vs. 0.23±0.07, respectively).
Spermiogram of stallions verified a significant alteration in semen pH (p<0.005), normality (p<0.001), individual motility (p=0.08), tail abnormalities (p<0.001) and total sperm count per ejaculate (p<0.05) with age during the hot summer season. The youngest age group (5-6 years) had a low pH (6.57±0.08), high individual motility (62.50±5.20%), lower tail abnormalities (28.00±1.15%) and total sperm count per ejaculate (15.18±1.71×109). Aged stallions (15-20 years) showed high pH (7.63±0.12), low individual motility (45.00±2.89%), low rate of normality (36.67±1.45%), high tail abnormalities (58.67±1.76%) and total sperm count per ejaculate (6.32±1.01×109).
3.2. Changes in lipid and protein profile in stallions of different ages during summer season
Estimation of lipid and protein profile declared a significant (p<0.05) difference in blood cholesterol level between stallions of different ages during hot summer (Table 2). Mean triglycerides and protein levels were not different between the studied stallion groups. Stallion of middle and old ages had a significant higher cholesterol level as compared with young aged group (86.95±12.61 and 92.60±7.69 vs. 58.53±4.52 mg/dL, respectively).
3.3. Changes in oxidative stress biomarkers in stallions of different ages during summer season
Evaluation of markers of oxidative stress in stallions declared a significant (p<0.05) difference in the total anti-oxidant capacity between studies stallion groups, though SOD, GSH and MDA was not statistically verified (Table 3). Total anti-oxidant capacity significantly (p<0.05) was higher in aged group (15-20 years) than young and middle age group (0.25±0.07 vs. 0.10±0.02 and 0.10±0.03 Mm/L, respectively).
3.4. Changes in serum micro- and macro-elements in stallion of different ages during summer season
Analysis of plasma micro and macro-elements levels in stallions declared noticeable variances among stallion examined groups with special emphasis to phosphorus (p=0.06) and sodium (p<0.05) levels, even though numerical differences existed in the other elements (Table 4). Young age group had a significant lower phosphorus (0.97±0.04 mmol/L) but higher sodium (172.75±8.68 mmol/L) than old age group (1.26±0.01 and 131.42±12.64 mmol/L, respectively).
3.5. Correlation between semen characteristics and lipid profile, oxidative stress markers and mineral levels in stallions during their breeding season
Data regarding the relation (Pearson-correlation coefficient) between semen characteristics and testosterone hormone, oxidative stress markers, lipid and protein levels, minerals in Arabian stallions during the summer hot season is presented in table 5.CAT was positively correlated with individual motility (r=0.83, p<0.001). SOD was negatively correlated with semen volume (r=-0.63, p<0.05). MDA was negatively correlated with semen pH and tail abnormalities (r=-0.78 and -0.68, p<0.05, respectively), but positively correlated with sperm normality and head abnormalities (r=0.69 and 0.68, respectively). TAC was positively (p<0.05) correlated with semen pH and tail abnormalities (r=0.77 and 0.67, respectively), but was negatively correlated with spermatozoa normality (r=-0.66, p<0.06).
Cholesterol level was positively (p<0.05) correlated with semen pH and tail abnormalities (r=0.07 and 0.73, respectively), but negatively (p<0.05) correlated with sperm normality (r=-0.60).
Cupper level was positively correlated with semen volume and sperm cell concentration (r=0.62 and 0.69, p<0.05, respectively). Zinc level was positively correlated with semen volume (r=0.63, p<0.05), but was negatively correlated with sperm livability (r=-0.77, p<0.05). Phosphorus level was negatively correlated with individual motility and normality (r=-0.66 and -0.64, p<0.05, respectively), but positively correlated with livability (r=0.79, p<0.06). Sodium level was negatively correlated with semen pH (r=-0.67, p<0.05), but positively correlated with sperm normality (r=0.63, p<0.05).
4. Discussion
Stallions may be afflicted by a long and varied list of differentials that related to physiologic, pathologic and management processes impact a stallion’s fertility. In the current study, testosterone (p=0.07), semen pH (p<0.005), normality (p<0.001), individual motility (p=0.08), tail abnormalities (p<0.001) and total sperm count per ejaculate (p<0.05) significantly differed between stallions of different ages and this was associated with significant differences in cholesterol (p<0.05), total anti-oxidant capacity(p<0.05), phosphorus (p=0.06) and sodium (p<0.05) levels.
Testes are responsible for the secretion of androgens and production of spermatozoa. Testosterone hormone is essential for the maintenance of normal spermatogenesis (Goeritz et al., 2003) and reproductive tract function (Luke and Coffey, 1994). Current data showed the testosterone level (p=0.07) as well as spermiogram (semen pH (p<0.005), normality (p<0.001), individual motility (p=0.08), tail abnormalities (p<0.001) and total sperm count per ejaculate (p<0.05)) significantly differed between stallions of different ages. Findings related to testosterone level agreed with McDonnell and Murray (1995) who found that the plasmatic testosterone level of harem stallions is higher than those of bachelor stallions in free-running horses. In the meantime, the spermiogram of young aged stallions herein characterized by high semen volume, total sperm count per ejaculate, motility and normality, but lower pH and tail abnormalities. These results indicated that the testosterone level could not be appropriate predictive of stallion fertility, though it is a main controller of the sex drive and sexual behavior. Roser and Hughes (1992) suggested that circulating plasma levels of FSH and estrogens, and not LH and testosterone, may be good markers to predict future changes in fertility. Inoue et al. (1993) suggested that the measurement of serum levels of estrogens and testosterone may help in the diagnosis of infertile stallions when other methods are not available. In latitudes higher than 30°, photoperiod is the most important cue regulating seasonal reproduction (Bronson and Heideman 1994). This suggested that the alteration in semen parameters might reflect the response of stallions of different ages to the high temperature of the summer breeding season. Leme et al. (2012) indicated the lack of seasonal differences in the testicular volume and semen parameters of tropical stallions, while Janett et al. (2003) showed that the volume of semen, sperm count and spermatozoa motility of fresh semen varied between summer and winter seasons. This difference might be due to the small variation in duration of natural light between the observed periods (Leme et al., 2012).
Studies on cholesterol, triglycerides and total proteins in domestic animals have clearly shown that species dissimilarities exist and that even within species significant differences occur. In the current data, while cholesterol differed notably with age in stallions during summer, triglycerides and total proteins were not varied. The values presented were obtained from clinically normal and healthy stallions. These findings could be mediated by thyroid gland activity. All aspects of lipid metabolism, including synthesis, mobilization and degradation are influenced by thyroid hormones, but degradation is affected more than synthesis. In mammals, thyroid hormone depletion leads to elevated serum cholesterol (Gueorguieva and Gueorguiev, 1997). There are contradictory findings regarding the relation between serum thyroid hormones and cholesterol and triglycerides. Former studies indicated that the age significantly increased cholesterol, triglycerides, total lipid, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol concentrations in Turkoman (Nazifi et al., 2003) and miniature (Nazifi et al., 2005) horses, while Mayer Valor et al. (1984) declared the non-significant effect of age on total lipids and cholesterol in Spanish horses. The differences could be attributed to the type of feeding program and the exercise of horses which need glucose consumption for energy (Hambleton et al., 1980).
A vast literature exists on the role of oxidative stress in the development of chronic pathological conditions and even aging in general. Ambient temperature and humidity have also been shown to influence oxidative stress. Measures of antioxidants can either focus on estimating the overall antioxidant capacity or the precise determination of individual key antioxidants. Data recorded herein verified statistical variation in the total anti-oxidant capacity between stallions of different age during the summer. In the mean time, numerical differences in other anti-oxidant markers were also found in this study. Low total antioxidant capacity could be indicative of oxidative stress or increased susceptibility to oxidative stress (Young, 2001). High liability to oxidative stress occurs due to an imbalance in antioxidants or the increased exposure to oxidants from the environment or increased production within the body from an increase in oxygen metabolism during exercise (McBride and Kraemer, 1999).Concurrently, former study showed that the serum nitric oxide concentrations of moderate and old horses were significantly lower than that in young horses (El Sisy et al., 2016).These results suggest that evaluating the antioxidant status and oxidative stress might prelude to the unpredicted low fertility of young stallions during hot breeding season.
There are some studies which demonstrate the significance of trace elements in male fertility. Increased levels of metal ions in blood plasma or semen appear to be significantly and positively correlated with male infertility (El Sisy et al., 2016). Successful reproduction requires complete provisions of micro- and macro-elements, including copper (Tuormaa, 2000), calcium and magnesium (Wong et al., 2001).In the current data, while plasma phosphorus decreased and sodium levels increased in stallion with age during the hot summer season.80% of Phosphate, one of the most abundant minerals in the body, present in bones and teeth, beside its importance for energy store. Sodium makes the principal base of plasma and its function appears to be physiochemical in nature, wherein it is responsible to maintain osmotic pressure and acid base balance. Factors contribute to the variation in macro-mineral concentrations during the physiological status include age, nutrition, breed (Durie et al., 2010) and horse blood-type (Mikniene et al., 2014). Moreover, Yamada et al. (1996) reported that exercise induced an increase in sodium and phosphorus retention due to a decrease in urinary excretion. Former studies indicated that the seasonal heat stress has profound effects on some serum biochemical parameters (Bengoumi et al., 1997) as a response to the climatic conditions and differences in the composition and characteristics of the diet (Bertoni, 1996). Vranković et al. (2015) assume that seasonal changes in mineral concentrations due to the greater muscle activity of the horses, especially in the summer period. Also, they suggest the possible influence of feed intake with regard to the way the animals are kept.
From these results it could be concluded that the age markedly distress stallion fertility, expressed by hormonal secretion and semen quality. Such effect might be mediated through influencing the metabolic (cholesterol level) status, oxidative stress resistance (total anti-oxidant capacity) and macro-minerals (phosphorus and sodium) during hot summer breeding season.