Influence of temperature–humidity index and seasonal variations on semen quality, scrotal circumference, and hormones in Kail rams raised under subtropical climate

Animals and location

Adult Kail rams (n = 5; age, 2–3 years; BCS, 3.5 ± 0.1) were used in this study. The Kail is a medium-sized sheep breed native to the Neelam and Leepa valleys of Azad Jammu and Kashmir (AJK), Pakistan. Adult rams average 41 kg in bodyweight and are characterized by a predominant white coat, with occasional black or brown head markings, medium ears, and a Roman nose. The breed is dual-purpose, raised for both meat and wool (fiber diameter, ~31 μm). All rams were housed at the experimental station of the University of Poonch (33.8584°N, 73.7654°E), Rawalakot, AJK, under a semi-intensive system. This included 6 h of grazing and access to water ad libitum. In winter, rams were provided hay feed alongside grazing and 400 g of concentrate consisting of soybean meal, canola meal, corn gluten and kernels, wheat bran, and maize grain. The Ethical Review Committee of the University of Veterinary and Animal Sciences, Lahore-Pakistan, approved all experimental protocols.

Meteorological data records

Monthly average of day length, relative humidity, temperature, THI, and day length from January to December 2022 are shown in Table 1. Seasons were defined as follows: winter: November to February; spring: March to 15 May; summer: 16 May to August; and autumn: September to October.

By using a previously published formula (Urie et al. 2018), THI was determined as follows:

where RH is the relative air humidity (%) and T is the dry bulb air temperature (°F).

Potential heat stress faced by the rams was assessed using the model outlined previously, which classifies THI values as follows: <72 (no stress), 72–78 (mild stress), 79–88 (moderate stress), and 89–98 (severe stress) (Armstrong 1994).

Semen processing and assessment

Semen from each ram (n = 5) was collected three times a month at 10-day intervals throughout 2022 via artificial vagina method (42°C). After measuring the volume of each ejaculate in a graduate collection vial, ejaculates of all five rams were pooled to mitigate individual variations. In total, 36 pooled ejaculates (3 pooled ejaculates per month × 12 months) were evaluated during this study. A computer-assisted sperm analyzer (CASA; SCA®, version 5.1, Microptic S.L., Spain) was used to evaluate the kinematics and motility of the sperm. In accordance with the manufacturer’s instructions, the CASA system made use of parameters that had already been verified (Hameed et al. 2023). These included the particle area: 20–60 μm², with a drift of 5 μm/s for slow, 10 μm/s for medium, and >25 μm/s for rapid sperm motion at 37°C. A connectivity threshold of 10 was applied to identify a valid particle. Sperm total motility (TM), progressive motility (PM), rapid progressive (RP), and medium progressive (MP) motility were analyzed. Sperm motion kinematics including curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF) were also analyzed. At least five different fields were observed and 500 sperm were assessed by placing a 5 μL drop of pooled semen on a pre-warmed (37°C) glass slide.

Blood collection and hormonal assays

To estimate plasma concentrations of testosterone and melatonin, monthly blood samples from five Kail rams were collected in EDTA vacutainers. Blood samples were centrifuged for 15 min at 3000g to harvest plasma. All samples were stored at −20°C until further analysis.

Plasma testosterone concentration was determined using a double-antibody radioimmunoassay (Immunotech, Beckman Coulter®, Czech Republic). With a minimum sensitivity of 0.04 ng/mL, the intra-assay and inter-assay coefficients of variation were 10.5% and 19% respectively. Melatonin plasma concentrations were measured using an enzyme-linked immunosorbent assay (ELISA)-based goat specific kit (KS18271, China) (Abbas et al. 2021). The intra-assay and inter-assay coefficients of variation in melatonin assay were 7% and 10% respectively.

Scrotal circumference (SC) and testicular echogenicity (TE)

Scrotal circumference (SC) was measured monthly in five rams over a period of 12 months by using a flexible scrotal tape (Barth tape). The testes were gently pulled to the bottom of the scrotum by grasping from neck and aligned side by side. The tape was snugly placed around the widest point of the scrotum without applying excess pressure and measurements were recorded in centimeters to ensure consistency and accuracy. Testicular echogenicity (TE) of both testes in each ram was assessed monthly for 12 months by using real-time B-mode ultrasound with a 7.5 MHz linear transducer (DRAMINSKI iScan®, Poland). Testicular ultrasonography was performed with the ram restrained in a standing position without sedatives. The transducer was placed parallel to the longitudinal axis of the testicle, scanning both the right and left testicles separately. The mediastinum of the testis served as a hyperechoic landmark for identifying the desired section of testicular parenchyma for pixel intensity (PI) calculation.

All testicular images were recorded at the same depth and focal point and then exported to a computer for further analysis. PI of each testicular sonogram was computed via Image J® software (version 1.46r; see https://ij.imjoy.io, National Institutes of Health, MD, USA) (Amjad et al. 2021). Six randomly placed squares of 2 mm size were selected on the testicular parenchyma, excluding the mediastinum. The PI of the image was calculated by averaging the estimates in six squares.

Statistical testing

All variables are expressed as means ± s.e.m. The influence of season and month on semen volume, quality, SC, TE, and plasma concentrations of testosterone and melatonin was analyzed through one-way ANOVA. To compare the months and seasons, Bonferroni’s posthoc test was employed. The association of THI with semen quality, hormones, and TE was evaluated using a linear regression model. The relationship between testicular echogenicity, and SC with semen picture and plasma concentration of testosterone and melatonin was analyzed using Pearson’s correlation coefficient. Statistical package SPSS (ver. 20.0, IBM Corporation, Armonk, NY, USA) was used to analyze the data, and statistical significance was considered at P ≤ 0.05.

Ethics approval

Animal Care and Ethical Review Committee of the University of Veterinary and Animal Sciences, Lahore-Pakistan approved the procedures (DAS 2037/29-11-2021).

Seasonal and monthly variations in semen volume and SC

Semen volume peaked in autumn and summer (1.2 ± 0.15 and 1.15 ± 0.09 respectively), with the lowest value observed in winter (0.73 ± 0.01). Scrotal circumference followed a similar trend, being significantly larger in summer (34.1 ± 0.38) than in other seasons, with the smallest values recorded in winter (26.6 ± 0.51; Table 2). The influence of months on semen volume and SC is shown in Table 3. Semen volume remained statistically stable (P > 0.05) from January to May. It increased (P < 0.05) from June through September compared with the January–May interval, then declined (P < 0.05) from October to December, returning to levels statistically similar to those observed earlier in the year. Scrotal circumference showed no significant (P > 0.05) change from January to May, then increased (P < 0.05) during June to August compared with January–March and December). Values declined from September onward, with those in November and December being significantly lower than those in the summer peak.

Seasonal and monthly variation in sperm motility and kinematics

Significant variations were noted in sperm motility parameters, including TM, PM, RP, and MP sperm, across the seasons and different months. Sperm TM and PM percentages were reduced significantly (P < 0.05) in winter compared with other seasons (Table 2). The TM and PM of spermatozoa were significantly higher during March–May and August–September than in January and December (Fig. 1a, b). The percentages of RP and MP sperm were highest in autumn and significantly lower in winter (Table 2). The RP sperm percentage was significantly higher during July–October than in November–March (Fig. 1c). The percentage of MP sperm was also significantly (P < 0.05) higher during April–May and August–September than during November–February (Fig. 1d).

Fig. 1.

Monthly variations of (a) motility, (b) progressive motility (PM), (c) rapid progressive (RP), and (d) medium progressive (MP) sperm percentage of Kail rams (n = 5). Different letters indicate significant differences at P = 0.05 between different months. Data are means ± s.e.m.

Among kinematic parameters, VCL, VAP, and VSL were significantly elevated in summer and autumn, whereas winter showed the lowest values. (Table 2). Sperm VCL values were significantly (P < 0.05) greater during April–October than November–February (Fig. 2a). Likewise, VAP values were significantly (P < 0.05) higher during May and July–September than in November–March (Fig. 2b). Sperm VSL values were higher during August through September than in November to March (Fig. 2c). Sperm ALH values were significantly lower in winter. Monthly analysis showed significantly (P < 0.05) higher ALH values during May and September than November to February (Fig. 2d). Likewise, sperm BCF values were lowest in winter and highest in summer. On a monthly basis, sperm BCF values were significantly greater during August–September than during November–March (Fig. 3a).

Fig. 2.

Monthly variations of sperm kinematics of Kail rams (n = 5). (a) Curvilinear velocity (VCL), (b) average path velocity (VAP), (c) straight-line velocity (VSL), and (d) amplitude of lateral head displacement (ALH). Different letters indicate significant differences at P = 0.05 between different months. Data are means ± s.e.m.

Fig. 3.

Monthly variations of (a) beat cross frequency of sperm (BCF), (b) plasma testosterone concentration, (c) melatonin concentration, and (d) testicular echogenicity of Kail rams (n = 5). Different letters indicate significant differences at P = 0.05 between different months. Data are means ± s.e.m.

Seasonal and monthly variation in testosterone and melatonin concentrations, and TE

No significant influence of seasons and months was observed on plasma concentrations of testosterone and melatonin (Table 2; Fig. 3b, c). TE values did not differ significantly among November, December, January, February, June, and July. The lowest values were observed in September, which were significantly lower than those in most other months (Fig. 3d). TE was significantly higher in winter (137.5 ± 3.2) than in autumn (102.5 ± 12.5), with spring (120.0 ± 5.8) and summer (130.0 ± 5.0) showing intermediate values that did not differ significantly from those of either winter or autumn (Table 2).

Effect of THI on sperm quality, hormonal profile, and TE

Regression analysis showed a significant (P < 0.05) positive association between THI and semen volume (R² = 0.70; Fig. 4a), TM (R² = 0.62; Fig. 4b), PM (R² = 0.65; Fig. 4c), RP sperm (R² = 0.68; Fig. 4d) and VCL (R² = 0.83; Fig. 5a). In contrast, THI had no significant association with plasma concentration of testosterone (R² = 0.02; Fig. 5b) and melatonin (R² = 0.08; Fig. 5c). A significant negative association of THI and TE was found (R² = 0.47; Fig. 5d).

Fig. 4.

The association of temperature–humidity index (THI) with (a) semen volume, (b) motility, (c) progressive motility (PM), and (d) rapid progressive sperm percentage (RP) of Kail rams (n = 5). y, dependent parameter; x, THI; R², coefficient of regression/determination.

Fig. 5.

The association of temperature–humidity index (THI) and (a) curvilinear velocity (VCL), (b) testosterone concentration, (c) melatonin concentration, and (d) testicular echogenicity of Kail rams (n = 5). y, dependent parameter; x, THI; R², coefficient of regression/determination.

Relationship between TE, SC, sperm motility and kinematics, testosterone, and melatonin

TE negatively correlated with TM, PM, and MP sperm. Likewise, TE had a negative correlation with VCL (r = −0.49), VAP (r = −0.43), ALH (r = −0.50), and BCF (r = −0.48). Non-significant correlations were found between TE, RP sperm (r = −0.25), VSL (r = −0.25), plasma concentration of melatonin (r = −0.26), and SC (r = −0.22). In contrast, TE had a significant negative correlation with plasma concentration of testosterone (r = −0.35; Figs 6a–l).

Fig. 6.

The relationship of (a) total motility, (b) progressive motility (PM), (c) rapid progressive sperm (RP), (d) medium progressive sperm (MP), (e) curvilinear velocity (VCL), (f) average path velocity (VAP), (g) straight-line velocity (VSL), (h) amplitude of lateral head displacement (ALH), (i) beat cross frequency (BCF) of sperm, (j) scrotal circumference (SC), plasma concertation of (k) melatonin and (l) testosterone, and testicular echogenicity of Kail rams (n = 5).

Relationship between SC, sperm motility and kinematics, testosterone, and melatonin

A significant positive correlation was found between SC and TM (r = 0.38), PM (r = 0.55), RP (r = 0.50) and MP sperm (r = 0.50), VCL (r = 0.58), VAP (r = 0.61), VSL (r = 0.61), ALH (r = 0.50), and BCF (r = 0.57). Correlations between SC and plasma concentrations of testosterone (r = 0.07) and melatonin (r = 0.12) were not significant (Figs 7a–k).

Fig. 7.

The relationship of (a) total motility, (b) progressive motility (PM), (c) rapid progressive sperm (RP), (d) medium progressive sperm (MP), (e) curvilinear velocity (VCL), (f) average path velocity (VAP), (g) straight-line velocity (VSL), (h) amplitude of lateral head displacement (ALH), (i) beat cross frequency (BCF) of sperm, and plasma concentation of (j) testosterone and (k) melatonin, and scrotal circumference of Kail rams (n = 5).