Heat stress impact on reproductive and physiological performance of Barbados Blackbelly rams under different management systems in the tropics

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Twenty-four Barbados Blackbelly rams were divided into intensive (ITS) and semi-intensive (SIS) management systems. ITS rams were housed in a barn, while SIS rams spent six hours daily outside over two months. Daily climate data were collected, and the Temperature-Humidity Index (THI) was calculated. Weekly semen collections and physiological assessments were conducted. Sperm motility was evaluated using a Computerised Assisted Sperm Analyser (CASA), alongside sperm viability, morphology, hormone levels, and oxidative stress. SIS rams experienced more severe heat stress, indicated by higher physiological responses ( p < 0.05) than ITS rams. SIS sperm quality was significantly lower, with reduced motility, progressive motility, kinematic parameters, morphology, and viability ( p < 0.05). Testosterone and cortisol levels showed no significant differences between systems, though ITS had higher testosterone and SIS had elevated cortisol. MDA ( p < 0.001) and H 2 O 2 ( p < 0.05) followed similar trends, with SIS rams showing higher oxidative stress levels. The THI for SIS was significantly greater than that of ITS, potentially affecting adaptable breeds like Barbados Blackbelly, resulting in lower fertility in SIS rams. Despite exceeding the THI threshold, the minimal impact on physiological responses in ITS rams suggests a contradiction, indicating a need for a specific classification of heat stress severity, with a focus on humidity in the tropics. Although heat stress (HS) impacts reproductive performance, physiological functions, and oxidative stress parameters, it does not significantly lead to infertility, and the rams generally remain fertile. Heat stress reproductive performance sperm quality hormones oxidative stress physiological responses Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Climate change poses a major threat to the livestock industry, particularly by contributing to heat stress occurrences among animals, including small ruminants such as sheep. Heat stress arises when the body temperature surpasses the thermoneutral level, prompting the activation of various physiological and behavioural adjustments in animals, including elevated rectal temperature, pulse and respiration rates (Mascarenhas et al. 2023 ; Pérez et al. 2020 ; Slimen et al., 2019 ). The Temperature-Humidity Index (THI) has been used to measure the discomfort levels endured by animals. It is regarded as a reliable method for assessing heat stress in animals, with different formulae that can be adapted to different geographical areas and animal species (Lallo et al. 2017; Thom 1959 ). Exposed to consistently high temperatures and humidity all year round, livestock in tropical regions face exacerbated challenges. It has been shown that the animals in these areas are prone to exposure to heat stress compared to those in other regions (Serrano et al. 2022 ). When exposed to heat stress, various physiological responses are triggered to counter elevated temperatures beyond the upper limit temperature (UCT) and maintain normothermia conditions (Schütz et al. 2024 ; Pérez et al. 2020 ). These responses include increased respiration rates, water consumption, body, and rectal temperature (Pulido-Rodríguez et al. 2021 ; Marai et al. 2007 ) to dissipate heat from the body. The adverse effects of heat stress extend beyond impacting compensatory activities in animals, leading to detrimental effects on growth and reproductive performance (Rebez et al. 2023 ). Although some experts believe that wool sheep would be exposed to more severe heat stress, hair sheep are also prone to the same exposure (McManus et al. 2020 ). Moreover, the decline in sheep reproductive performance attributable to heat stress is also manifested through sperm quality, whether in fresh or cryopreserved form, as well as modifications in the composition of ejaculated seminal plasma (Moula et al. 2024 ). A breed’s inherent adaptability to adverse environmental conditions plays a crucial role in mitigating the impacts of heat stress, thereby preserving overall productivity. One example is Barbados Blackbelly, renowned for its adaptability to tropical climates and high productivity rates (Joy et al. 2020 ). While the breed is well-suited to thrive in tropical climates, it remains vulnerable to the detrimental effects of heat stress, particularly on reproductive performance resulting from elevated reactive oxygen species (ROS), which leads to a deterioration in semen quality (Moula et al. 2024 ; De et al. 2017 ). The reproductive performance deterioration during heat stress contributed to the accumulation of reactive oxygen species (ROS) and later increased the incidence of oxidative stress (OS). Higher OS concentration exceeding the normal balance affects the activity of antioxidants in the cell, which later exposes cells such as spermatozoa to OS, resulting in a significant loss in sperm quality (Lekola et al. 2024 ). Heat stress resulting from environmental stress could induce metabolic responses that enhance OS through the elevation of ROS in the cells, such as hydrogen peroxide, superoxide anion, and radical hydroxyl (El-Zeftawy et al. 2020 ). To reduce the effects of heat stress exposure on small ruminants, several management systems are practised in tropical countries, such as an intensive system that confines the animals and applies specific dietary regulations with a focus on high productivity (Moorby & Fraser 2021 ; Simoes et al. 2021), as well as a semi-intensive system that involves diurnal grazing and nocturnal confinement with a regulated dietary routine suitable for their growth and reproduction compared to free-range systems (Shivakumara & Kiran 2019 ). Direct exposure to adverse environments could lead to heat stress occurrence. Therefore, animals managed semi-intensively are more likely to be impacted (Jack et al. 2022 ). Furthermore, THI measurements are different for both management systems and could lead to heat stress of varying severity. Therefore, this study aimed to investigate how different THI in different small ruminant management systems in a tropical environment induce heat stress and impact rams’ fertility, emphasising the physiological responses and sperm quality of Barbados Blackbelly rams. MATERIALS AND METHODS a) Animals and Management Systems This study used twenty-four Barbados Blackbelly rams aged between two and three years old with body weights of between 48 and 60 kg. The animals were fed with commercial pellets and chopped Napier grass daily with ad libitum drinking water. The rams were randomly divided into two groups and placed into one of two types of management system: intensive (ITS), which involved a single pen inside elevated slatted flooring pens, and semi-intensive (SIS), in which the animals could range freely outside the animal housing with no shade provided. The SIS rams were kept outside within the designated fence area, exposed to optimum sunlight with ad libitum water. Rams in the semi-intensive system were allowed to range for six hours daily from 9:00 am to 3:00 pm over a two-month period. b) Location The study was conducted at the Glami Lemi Biotechnology Research Centre Universiti Malaya, Jelebu, Negeri Sembilan, Malaysia (3° 3' 50.2452'' N; 102° 3' 26.7588'' E). The sheep barn and the weather station were about 100 metres apart. c) Climate Data The study was conducted in three cycles within 2022 – 2024, between February and August. The temperature and humidity data were measured by using a Temperature-Humidity logger (Elitech GSP-6) for the intensive system (hanging on the top of the experimental ram’s pen). Meanwhile, the Temperature-Humidity Index (THI) for the animals under semi-intensive management was evaluated using a weather station, Rainwise® MKIII. The logger and the weather station recorded the temperature and humidity every 30 minutes daily for two months. Measured data were retrieved from 9:00 am to 3:00 pm with 30-minute intervals. THI values were calculated using the formulation below: THIs = 3.43 + (1.058) (Ta) – 0.293 × RH + 0.0164 × Ta × RH + 35.7 (Berman et al., 2016) THI = Temperature-Humidity Index Ta = Ambient temperature, °C RH = Relative humidity as a fraction of the unit, % d) Physiological Assessment Physiological parameters such as rectal temperature (RT) (measured using a rectal thermometer), heart rate (HR) per minute (a CMS60D-VET pulse meter), respiration rate (RR) per minute (flank movement), and testicular temperature (TT) (an infrared thermometer) were assessed weekly after 3:00 pm. To reduce the effect of vigorous movement during the assessment procedures, the rams were kept calm for five minutes before any assessment. e) Sperm Quality Semen was collected using an artificial vagina (AV) weekly at 3:00 pm for two months. The semen was diluted with a Tris-Citrate Acid Yolk Extender (TCAYE) before sperm kinematics and viability characteristics evaluations were conducted using a Computerised Assisted Sperm Analyser (CASA), IVOS (Hamilton Thorne). The semen was diluted 100- and 10-fold for the kinematic and viability evaluations, respectively. About 10 µL of diluted sperm was placed onto a Makler chamber, and three technical replicates were recorded for each sample. Sperm kinematic parameters from the intensive and semi-intensive systems were evaluated using IVOS (Animal motility module) version 12, with the following settings: 30 frames at a frame rate of 60 frames/s; minimum contrast = 20; minimum cell size = 5 pixels; motility = 30 μm/s; progressive motility = 50 μm/s; straightness = 70%. The Makler chamber was heated to 37 °C prior to the sample observation. The kinematic-related variables evaluated were the VAP (velocity average path), VCL (curvilinear velocity), VSL (straight-line velocity), BCF (beat cross frequency), ALH (amplitude of lateral head displacement), total and progressive motility, and percentage of cells with rapid, medium, slow, and static movements for the sperm velocity distribution. Sperm viability and abnormalities (head, midpiece, and tail) were assessed using premixed eosin-nigrosin staining (Nidacon®). A total of 200 sperm were counted with three technical replicates for each sample. The sperm quality were also measured at 1, 2, and 4 hours after incubation in a water bath at 37 °C for thermoresistance test (Lange-Consiglio et al., 2013). f) Hormone and oxidative stress levels Enzyme-linked Immunosorbent Assay (ELISA) was conducted to detect testosterone, cortisol and malondialdehyde (MDA) levels using pre-coated FineTest Ⓡ ELISA plates. Hydrogen peroxide (H 2 O 2 ) was analysed using CheKine TM Micro colourimetric assay. Hormone levels were evaluated using blood serum, while oxidative stress markers were evaluated using seminal plasma. g) Statistical analysis Data were analysed by one-way repeated measures ANOVA according to incubation time and week parameters under the different management systems and THI. Student’s t-test was used to evaluate the differences between the means of the two treatment groups. All statistical analyses were conducted at a significance level of p <0.05 or p <0.001 using IBM SPSS software version 29. Data were expressed as means and standard error. RESULTS Temperature-Humidity Index (THI) The THI in the SIS was significantly higher ( p < 0.05) (Figs. 1 and 2 ) compared to that in the ITS. Both management systems entered the danger range of the THI at 9:00 am. The SIS reached an emergency THI range earlier than the ITS, indicating that the SIS rams were exposed to severe discomfort earlier and longer than their ITS counterparts. Despite that, the Pearson correlation test showed a positive correlation between the THI of both ITS and SIS ( r = 0.531, p < 0.05), Physiological responses The discomfort level, as reflected in all the physiological parameters, is shown in Table 1 . The SIS rams recorded significantly higher ( p 0.05). In contrast, the weeks (Fig. 3 ) did not influence the physiological responses in either the ITS or SIS rams ( p > 0.05). TT of the SIS rams at weeks 1, 4, 5, 7, and 8 (Fig. 3 ) showed no significant differences with the TT of the ITS rams, although the SIS rams had higher TT. The Pearson correlation test found the THI in the ITS was not correlated with the HR ( r = -0.026, p > 0.05), RT ( r = -0.124, p > 0.05), or TT ( r = -0.172, p > 0.05), except RR which showed a significant correlation ( r = 0.285, p < 0.05). Meanwhile, significant correlations were recorded between the THI and the HR ( r = 0.207, p < 0.05), RT (r = 0.302, p < 0.05), TT ( r = 0.172, p < 0.05), and RR ( r = 0.217, p < 0.05) for all physiological assessments of the rams in the SIS, Table 1 Physiological parameters of rams in different management systems Parameter Management System ITS (n = 12) SIS (n = 12) Heart rate (beats/minute) 80.03 ± 1.49 114.29 ± 2.23 * Respiration rate (breaths/minute) 86.13 ± 2.43 135.58 ± 3.28 * Rectal temperature (°C) 38.54 ± 0.05 39.30 ± 0.04 * Testicular temperature (°C) 36.38 ± 0.03 36.54 ± 0.02 * Values are expressed as mean ± S.E. * indicates a significant difference in the same row using T-test ( p < 0.05). Sperm quality The current study recorded no significant differences between the management systems’ semen volume and sperm concentration of the BB rams (Table 2 ). The sperm viability of the ITS rams was significantly better than that of the SIS rams. Meanwhile, in relation to sperm abnormality (Table 3 ), the head, midpiece, and tail abnormalities of the sperm of the SIS rams were significantly higher ( p < 0.05) than those of the ITS rams. This was also evident in the increased susceptibility of the sperm to abnormalities and the more rapid decline in sperm quality with prolonged incubation, compared to rams maintained under the ITS. (Table 4 ). Table 2 Semen volume and sperm concentration of rams in different management systems Parameters Management Systems ITS (n = 12) SIS (n = 12) Semen volume (mL) 0.98 ± 0.03 0.96 ± 0.04 Sperm concentration (10 9 /mL) 1.98 ± 0.06 2.04 ± 0.07 Values are expressed as mean ± S.E. * indicates a significant difference in the same row using T-test ( p < 0.05). Table 3 Sperm viability and morphology abnormalities of rams in different management systems Parameters Management Systems ITS (n = 12) SIS (n = 12) Viability, (%) 92.51 ± 0.27* 88.67 ± 0.41 Abnormality, (%) 3.50 ± 0.19 5.20 ± 0.25* Head abnormality, (%) 2.03 ± 0.06 2.74 ± 0.07* Midpiece abnormality, (%) 1.24 ± 0.03 1.71 ± 0.05* Tail abnormality, (%) 1.86 ± 0.05 2.46 ± 0.06* Values are expressed as mean ± S.E. * indicates a significant difference in the same row using T-test ( p < 0.05). Table 4 Sperm viability and morphology abnormalities of rams over different incubation times and under different management systems Parameter Incubation Time (h) Management systems ITS (n = 12) SIS (n = 12) Viability, (%) 0 (Fresh) 92.46 ± 0.39 b,z 88.67 ± 0.41 a,z 1 89.20 ± 0.40 b,y 85.87 ± 0.38 a,y 2 85.87 ± 0.37 b,x 83.05 ± 0.34 a,x 4 82.60 ± 1.43 b,w 78.21 ± 0.44 a,w Abnormality, (%) 0 (Fresh) 3.50 ± 0.19 a,w 3.72 ± 0.25 a,w 1 4.55 ± 0.22 a,x 6.07 ± 0.20 b,x 2 5.45 ± 0.20 a,y 7.36 ± 0.22 b,y 4 6.71 ± 0.20 a,z 8.72 ± 0.27 b,z Values are expressed as mean ± S.E. Superscript a,b indicates a significant difference in the same row within the same parameters using T-test (p < 0.05). Superscript w,x,y,z indicates a significant difference in the same column within the same management system using repeated measures ANOVA ( p < 0.05). Data presented in Table 5 shows that the sperm motility and progressive motility of the SIS rams were significantly lower ( p < 0.05) than ITS rams. The results revealed a significantly greater deterioration in sperm parameters of SIS rams compared to ITS rams after 4 hours of incubation. Other kinematic parameters indicated notable differences in the results regardless of the incubation time, with the VAP, VSL, and VCL recorded as being significantly higher ( p < 0.05) in the SIS rams (Table 6 ). Nonetheless, no notable differences in ALH and LIN were observed between the fresh readings and those taken after 1 hour of incubation hour. Meanwhile, no significant difference was found between the management systems for STR during 1 hour of incubation. Nonetheless, for incubation times of 2 and 4 hours, the kinematic parameters exhibited consistent significant trends similar to other kinematic parameters, with the SIS rams’ sperm recorded as significantly lower (p < 0.05) compared to their ITS counterparts for ALH, LIN, and STR. Table 5 Sperm motility and progressive motility over different incubation times and under different management systems Parameter Incubation Time (h) Management systems ITS (n = 12) SIS (n = 12) Total motility, (%) 0 (Fresh) 92.92 ± 0.40 b,z 87.18 ± 0.75 a,z 1 89.69 ± 0.57 b,y 82.50 ± 0.95 a,y 2 82.85 ± 0.87 b,x 66.33 ± 1.55 a,x 4 60.46 ± 1.86 b,w 38.94 ± 2.02 a,w Progressive motility, (%) 0 (Fresh) 43.93 ± 0.74 b,z 32.57 ± 0.72 a,z 1 22.59 ± 0.44 b,y 11.93 ± 0.48 a,y 2 15.23 ± 0.71 b,x 6.51 ± 0.46 a,x 4 7.62 ± 0.55 b,w 1.86 ± 0.22 a,w Values are expressed as mean ± S.E. Superscript a,b indicates a significant difference in the same row within the same parameters using T-testing ( p < 0.05). Superscript w,x,y,z indicates a significant difference in the same column within the same management system using repeated measures ANOVA ( p < 0.05). Table 6 Kinematic parameters over different incubation times and under different management systems Parameter Incubation Time (h) Management systems (Mean ± SE) ITS (n = 12) SIS (n = 12) Path velocity (VAP), µm/s 0 (Fresh) 107.04 ± 0.92 b,z 97.64 ± 1.00 a,z 1 88.84 ± 2.36 b,y 73.93 ± 1.21 ,a,y 2 67.74 ± 1.11 b,x 55.28 ± 0.1.08 ,a,x 4 50.92 ± 1.21 b,w 35.99 ± 1.43 a,w Progressive velocity (VSL), µm/s 0 (Fresh) 84.14 ± 0.84 b,z 74.56 ± 0.76 a,z 1 64.55 ± 0.58 b,y 54.40 ± 0.72 a,y 2 52.68 ± 0.70 b,x 42.95 ± 0.76 a,x 4 40.72 ± 0.98 b,w 27.51 ± 1.12 a,w Track speed (VCL), µm/s 0 (Fresh) 185.56 ± 1.83 b,z 172.60 ± 2.06 a,z 1 159.77 ± 2.07 b,y 135.92 ± 2.50 a,y 2 121.00 ± 2.24 b,x 101.23 ± 1.92 a,x 4 89.91 ± 2.10 b,w 66.81 ± 2.59 a,w Lateral amplitude (ALH), µm 0 (Fresh) 7.73 ± 0.07 a,z 7.81 ± 0.07 a,z 1 8.20 ± 0.26 a,z 7.61 ± 0.35 a,z 2 6.54 ± 0.13 b,y 5.40 ± 0.39 a,y 4 4.72 ± 0.32 b,x 2.62 ± 0.21 a,x Straightness (STR), % 0 (Fresh) 77.35 ± 0.39 b,z 75.67 ± 0.36 a,z 1 73.74 ± 0.43 a,y 74.56 ± 0.44 a,z 2 77.64 ± 0.45 b,z 75.70 ± 0.73 a,z 4 71.65 ± 1.32 b,y 56.39 ± 2.01 a,y Linearity (LIN), % 0 (Fresh) 46.54 ± 0.40 a,z 45.10 ± 1.08 a,z 1 41.41 ± 0.28 a,x 41.40 ± 0.33 a,y 2 43.73 ± 0.45 b,y 42.30 ± 0.42 a,y 4 40.64 ± 0.82 b,x 31.32 ± 1.15 a,x Values are expressed as mean ± S.E. Superscript a,b indicates a significant difference in the same row within the same parameters using T-testing (p < 0.05). Superscript w,x,y,z indicates a significant difference in the same column within the same management system using repeated measures ANOVA ( p 0.05), despite the testosterone concentration of ITS being slightly higher compared to SIS, indicating a trend due to the impact of the heat stress, especially for SIS., consistent with physiological and sperm quality parameters Concurrently, the cortisol concentration of SIS was slightly higher than that of ITS, despite no significant difference recorded in both systems. Oxidative stress indicators such as malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were significantly higher in SIS seminal plasma than ITS ( p < 0.001). Table 7 Hormones oxidative stress levels in different management systems Hormones ITS (n = 12) SIS (n = 12) Testosterone, ng/mL 160.57 ± 7.5 150.69 ± 6.9 Cortisol, ng/mL 235.28 ± 12.65 243.10 ± 18.40 Oxidative Stress Malondialdehyde, nmol/mL 1.25 ± 0.15 2.51 ± 0.17** Hydrogen Peroxide, µM 124.95 ± 0.74 133.31 ± 0.67** Values are expressed as mean ± S.E. **indicates a significant difference in the same row of the same treatment using T-test ( p < 0.001) DISCUSSION A higher THI was recorded in the SIS than the ITS, reflecting the ram experiencing discomfort. Referring to Thom (1957) and supported by Hahn et al. ( 2009 ) and Berman et al. ( 2016 ), the THI ranges of severity classification for ruminants are as follows: normal ≤ 74; moderate 75–78; severe 79–83; and very severe (emergency) ≥ 84. The ITS effectively regulates the THI in the immediate environment of the animals, which is especially important during extreme weather events when THI levels may spike significantly. The THI correlation of ITS and SIS, indicating that the climate conditions to which the SIS was exposed directly also affected the THI in the ITS. Physiological responses such as HR, RR, RT, and TT have been reported as reliable indicators of heat stress occurrence in small ruminants (Sejian et al. 2017 ; Al-Dawood 2007; Marai et al. 2007 ). These responses are part of the thermoregulatory countermeasures to mitigate excessive heat load in the body (Moula et al. 2024 ; Ghezzi et al. 2023; Li et al. 2018 ). The current study revealed that the SIS rams experienced greater heat stress compared to the ITS rams. A primary reason for this was the exposure of the SIS rams to solar radiation, leading to a higher radiant temperature compared to the air temperature observed in the ITS (Schütz et al. 2024 ; Silva et al. 2010 ). On the other hand, the ITS rams were also exposed to a very severe THI range based on Hahn et al. ( 2009 ) classification. However, their physiological responses, as shown in Table 1 , did not reflect stress behaviour, in contrast to the responses of the SIS rams. Previous reports indicated the physiological responses of Santa Ines rams under heat stress, which recorded a respiratory rate (RR) of 113 breaths per minute, a heart rate (HR) of 134 beats per minute, and a rectal temperature (RT) of 39.6°C (Gesualdi et al. 2014; Rashamol et al. 2020 ). Thus, the air temperature in the ITS was assumed not to affect the rams significantly, even though the THI exceeded the emergency threshold. This suggests that the THI range of classification was not ideal for the ITS in the tropics, and the fact that minimal effects were observed may be attributed to the breed-specific characteristics of the Barbados Blackbelly sheep, which showed no significant physiological responses despite the THI threshold for heat stress being surpassed. Furthermore, the adaptive traits of Barbados Blackbelly rams to tropical adverse environmental conditions may help mitigate the effects of heat stress (Paim et al. 2021 ; de Almeida et al. 2018; Horton & Burgher 1992 ). It was also reported by Maurya et al. ( 2019 ) that the effects of heat stress on sheep differ depending on their sex, where it has been reported that males exhibited better adaptability to heat stress than females. Females have greater susceptibility to heat stress, which is reflected in their severe physiological, endocrine and blood-biochemical responses than males (Maurya et al. 2019 ). The correlation of RR with THI in ITS is due its efficiency for physiological response that results in a more vigorous mechanism for eliminating excessive heat load from the body through respiratory tract evaporation, ensuring a homeothermic condition (Silva et al. 2024 ). Although the THI value recorded in the ITS exceeded the threshold designated for emergency conditions, the physiological responses detailed in Table 1 and Fig. 3 do not correspond with the anticipated severity of heat stress. This indicates that the THI range may not have substantially impacted the ITS rams' physiological status. This could be reflected in, for example, heart rates of 80 beats/minute in a resting state (Yaxley et al. 2021 ) and 90–95 beats/minute in a normal state (Shilja et al. 2016 ), which are equivalent with the data obtained for the ITS rams. This indicates that the ITS rams were not experiencing extreme heat stress, suggesting that the existing common THI ranges for heat stress severity may not be suitable for BB sheep in the tropics. According to Berman et al. ( 2016 ), the common THI classifications were mostly based on ambient temperature (Ta). This overemphasised the temperature diminished the significance of humidity in the animal heat stress, particularly in tropical regions, where humidity is more prominent. Because the THI index might not adequately reflect the combined effects of temperature and humidity, the animals might not show physiological stress reactions even though the THI readings fall into the emergency category (≥ 84). This is especially important in settings where humidity contributes significantly to heat stress but is not sufficiently considered when calculating THI. The significant correlation of SIS for all physiological parameters with the THI implying that they were exposed to more heat stress as the THI rose. The correlations suggest that the SIS rams displayed significant physiological responses to maintain normothermia conditions and regulate their body temperature when approaching the upper limit of their thermoneutral zone (Pérez et al. 2020 ; Schütz et al. 2024 ). These findings align with those of a previous study by Mascarenhas et al. ( 2023 ), who reported a correlation between physiological responses and THI. Furthermore, in this study, TT was consistently lower than the RR for the entire research period. This implied that testicular thermoregulation had occurred to maintain a TT below the body core temperature. Increased TT due to heat stress can have detrimental effects on the seminiferous tubule diameter (STD) and testicular weight, which could subsequently lead to detrimental effects on the epididymal sperm and spermatids (Teixeira et al., 2024 ). Severe heat stress can impair testicular function by inducing oxidative stress, thereby disrupting intracellular signal transduction (Lekola et al. 2024 ; Ahsan et al. 2014 ). The spermatogenesis cycle in rams lasts 49 days, leading to the storage of sperm in the epididymis prior to ejaculation. It has been reported that the deleterious effect of heat stress will not affect mature spermatozoa as its effect occurs mainly during earlier stages of sperm development (Sierra et al. 2021 ; Rizzoto & Kastelic 2020 ; El Amiri 2020). Thus, minimal changes were observed in the semen volume and sperm concentration under both management systems. Nevertheless, heat stress has been discovered to negatively influence sperm concentration in dorper rams during summer, indicating a period of high ambient temperature (Barragan et al. 2023). Based on the current study, most of the recorded sperm quality parameters indicated more negative impacts on the SIS rams compared to the ITS rams. Sperm morphology is impaired during heat stress due to detrimental changes in the meiosis cycle of the spermatocytes and spermiogenesis. Prolonged exposure to chronic heat stress exceeding two months could result in 3.6% of sperm developing major abnormalities and 43.4% developing minor abnormalities (Moula et al. 2024 ). Meanwhile, short exposure to heat stress could also lead to more sperm abnormalities due to sperm epididymal damage (Barragan et al. 2024). The effect of sperm morphology impairment after a brief exposure to heat stress may be observed after several weeks or as early as two weeks after the exposure, and the effects might persist for a minimum of eight days and last for up to 77 days (Shahat et al. 2020 ; Abdelhamid et al., 2019 ). Consequently, the effects may persist up to two months, although the exposure to heat stress has been diminished (Shahat et al. 2020 ). The data in Tables 2 and 3 also align with previous studies on boar sperm. The previous study reported that exposure to heat stress resulted in an increase in sperm abnormalities, with no effects on the semen volume or sperm concentration (Rizzota & Kastelic 2020; Li et al. 2015 ; Cameron & Blackshaw,1980). The sperm motility and progressive motility deteriorated severely after four hours of incubation, implying that exposure to heat stress under the SIS could increase sperm susceptibility. In addition, repeated measures analysis showed significant interactions ( p < 0.001) between the management system and the incubation time. This implies that the management system and incubation time both affect the total motility and progressive motility of the sperm. These new findings are also supported by previous studies and reviews by Barragán et al. ( 2023 ) and Moula et al. ( 2024 ), who reported a major decrease in the sperm quality of rams under heat stress. Meanwhile, in line with other kinematic parameters, the ITS rams exhibited significantly more rapid sperm than the SIS rams. It is critical to maintain sperm with rapid velocity as these have greater potential to penetrate the cervical mucus, increasing the potential for fertilisation, while slower sperm may hamper fertilisation (Johnson et al. 2020 ; Ford et al. 1992 ). The decrease in sperm thermoresistance in rams reared in the SIS could be attributed to the build-up of reactive oxygen species (ROS), leading to oxidative stress and lipid peroxidation during heat stress exposure. This cascade chemical reaction negatively affects sperm motility (Teymoori et al. 2024 ; Qamar et al. 2023 ; Dutta et al. 2019 ). Moreover, the downregulated of several genes related to the translation of carboxypeptidase Q-precursor ( CPQ ), superoxide dismutase ( SOD1 ) and NPC intracellular cholesterol transporter 2 ( NPC2 ) that are responsible in activation of hormones peptides and oxidative stress regulation as well as epidydimal transit functions were also reported to occur concurrently as the sperm motility in rams deteriorated during heat stress (Neto et al. 2024 ). Furthermore, the SIS rams were more affected by heat stress than ITS, which was also supported by physiological response results shown in Table 1 and Fig. 3 , where the RT, TT, HR and RR were significantly higher in SIS rams. The optimal kinematic parameters must be attained as these are responsible for determining animal fertility, especially VAP, which is highly correlated with animal fertility status (Tanga et al. 2021 ; Nagy et al. 2015 ). Thus, the current study indicates that sperm from an SIS may result in reduced ram fertility compared to sperm from an ITS. At first, both the STR and LIN parameters in the management systems decreased for an hour before starting to recover after 2 hours of incubation. The recovery observed could be attributed to the spermatozoa’s adaptability to the extender microenvironment, which has been reported to affect sperm functionality (Neila-Montero et al. 2024 ). Meanwhile, the VCL and STR were significantly higher in the ITS (p < 0.05) compared to the SIS. Higher VCL and STR are known to reflect higher sperm hyperactivation; this is crucial for zona pellucida penetration, which is a prerequisite in fertilisation (Suarez 2008 ). Furthermore, the VCL and VAP of the sperm have been reported to play critical roles in fertilisation due to their major attribute of transporting spermatozoa through the mucus of the ewe cervix (Van de Hoek et al. 2022 ). Furthermore, it is known that testosterone plays a critical role in maintaining spermatogenesis (Sharpe et al. 1981 ). The fact that the current study showed deteriorated sperm quality, especially for SIS, indicates an adverse effect of testosterone concentration during the early sperm maturation process. The elevated cortisol levels represent a component of the heat stress response observed in the SIS rams, which was triggered by the hypothalamic-pituitary-adrenal (HPA) axis and the sympatho-adrenal-medullary (SAM) system. This response leads to adrenocorticotropic hormone (ACTH) secretion, subsequently stimulating glucocorticoid production, including cortisol (Barragán Sierra et al. 2021 ). It could be interpreted that an increase in reactive oxygen species (ROS) due to heat stress in SIS led to oxidative damage through multiple pathways, such as the peroxidation of polyunsaturated fatty acids (PUFAs) affect the sperm membranes, DNA fragmentation, and impaired mitochondrial function, where the impacts collectively deteriorate sperm quality and ultimately affecting fertilisation capability. Moreover, heat stress amplifies ROS production and reduces antioxidant defences in seminal plasma, creating a synergistic effect that heightens the harmful influence of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) on sperm quality, leading to a decline in fertility potential (Agarwal et al. 2014; Durairajanayagam et al. 2015). The H₂O₂ impact includes irreversible axoneme damage responsible for protein production in the flagellum (Aitken et al. 2012). Despite having a higher MDA concentration than in ITS, the concentration of MDA in SIS was less than 3 nmol/L, indicating the MDA concentration was still within the fertile range based on Almady et al. (2021). The other parameters were also aligned with the MDA data, indicating that most of the SIS parameters were documented as being more severely affected by heat stress than those of the ITS. Still, it is noteworthy that the severity remained within an acceptable range and did not lead to infertility. In conclusion, exposing Barbados Blackbelly rams to a SIS reduces the effects of heat stress on their physiological, reproductive, and oxidative stress levels compared to an ITS. However, the impact does not extend to infertility in the rams. The negative effects on reproductive performance stay within an acceptable range. Thus, the semi-intensive system may remain in use, provided that effective mitigation measures are implemented to minimise the effects of heat stress. Declarations Ethical approval The use of animals in the study was approved by the Universiti Malaya Institutional Animal Care and Use Committee (UM IACUC) with reference number S/28062022/03062022-01/R. Conflict of interest statement: The authors declare that there is no conflict of interest. Author ORCID Mohd Shahmi Hakimi Mazlishah − 0000-0003-0221-3600 Nuradilla Mohamad-Fauzi − 0000-0001-5192-9889 Mohd Fadzil Firdzaus Mohd Nor − 0000-0002-6145-6381 Muhammad Syafiq Roslan − 0009-0007-7370-9247 Shamsul Azlin Ahmad Shamsuddin − 0000-0001-7983-6502 Noor Hashida Hashim − 0000-0003-2572-4083 Funding The study was funded by the Ministry of Higher Education (MOHE) under the Fundamental Research Grant Scheme (FRGS/1/2022/STG03/UM/02/7). Author contributions Mazlishah, M.S.H., wrote the first draft and carried out the experiment, data collection, optimisation, and correction. Hashim, N.H., reviewed the draft, supervised the study, and made the experimental design. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6779838","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468776362,"identity":"2467b05f-bc85-4bd8-823d-aa332ecd7ad9","order_by":0,"name":"MOHD SHAHMI HAKIMI 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systems.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6779838/v1/1291b824fac326adab890555.jpg"},{"id":84389269,"identity":"2162ca16-9cf4-440f-a989-de1391f950bc","added_by":"auto","created_at":"2025-06-11 10:55:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWeekly average Temperature-humidity index (THI) in different management systems\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6779838/v1/4bead99296636c25b4b93b5b.jpg"},{"id":84389480,"identity":"de1c6cc2-ac7b-4f43-96fa-49ed1dfddd88","added_by":"auto","created_at":"2025-06-11 11:03:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":281667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological responses across the weeks under different management systems, a) heart rate, b) respiration rate, c) testicular temperature, d) rectal temperature\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6779838/v1/89a5f9f877858d2f573afffb.jpg"},{"id":87479843,"identity":"e7a93997-18a6-469f-aea4-a514192512c3","added_by":"auto","created_at":"2025-07-24 09:41:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1378225,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6779838/v1/1abd8017-a1b6-412b-8c88-abd31b269965.pdf"}],"financialInterests":"","formattedTitle":"Heat stress impact on reproductive and physiological performance of Barbados Blackbelly rams under different management systems in the tropics","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eClimate change poses a major threat to the livestock industry, particularly by contributing to heat stress occurrences among animals, including small ruminants such as sheep. Heat stress arises when the body temperature surpasses the thermoneutral level, prompting the activation of various physiological and behavioural adjustments in animals, including elevated rectal temperature, pulse and respiration rates (Mascarenhas et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; P\u0026eacute;rez et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Slimen et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The Temperature-Humidity Index (THI) has been used to measure the discomfort levels endured by animals. It is regarded as a reliable method for assessing heat stress in animals, with different formulae that can be adapted to different geographical areas and animal species (Lallo et al. 2017; Thom \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1959\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExposed to consistently high temperatures and humidity all year round, livestock in tropical regions face exacerbated challenges. It has been shown that the animals in these areas are prone to exposure to heat stress compared to those in other regions (Serrano et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When exposed to heat stress, various physiological responses are triggered to counter elevated temperatures beyond the upper limit temperature (UCT) and maintain normothermia conditions (Sch\u0026uuml;tz et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; P\u0026eacute;rez et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These responses include increased respiration rates, water consumption, body, and rectal temperature (Pulido-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Marai et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) to dissipate heat from the body. The adverse effects of heat stress extend beyond impacting compensatory activities in animals, leading to detrimental effects on growth and reproductive performance (Rebez et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although some experts believe that wool sheep would be exposed to more severe heat stress, hair sheep are also prone to the same exposure (McManus et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the decline in sheep reproductive performance attributable to heat stress is also manifested through sperm quality, whether in fresh or cryopreserved form, as well as modifications in the composition of ejaculated seminal plasma (Moula et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA breed\u0026rsquo;s inherent adaptability to adverse environmental conditions plays a crucial role in mitigating the impacts of heat stress, thereby preserving overall productivity. One example is Barbados Blackbelly, renowned for its adaptability to tropical climates and high productivity rates (Joy et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While the breed is well-suited to thrive in tropical climates, it remains vulnerable to the detrimental effects of heat stress, particularly on reproductive performance resulting from elevated reactive oxygen species (ROS), which leads to a deterioration in semen quality (Moula et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; De et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The reproductive performance deterioration during heat stress contributed to the accumulation of reactive oxygen species (ROS) and later increased the incidence of oxidative stress (OS). Higher OS concentration exceeding the normal balance affects the activity of antioxidants in the cell, which later exposes cells such as spermatozoa to OS, resulting in a significant loss in sperm quality (Lekola et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Heat stress resulting from environmental stress could induce metabolic responses that enhance OS through the elevation of ROS in the cells, such as hydrogen peroxide, superoxide anion, and radical hydroxyl (El-Zeftawy et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo reduce the effects of heat stress exposure on small ruminants, several management systems are practised in tropical countries, such as an intensive system that confines the animals and applies specific dietary regulations with a focus on high productivity (Moorby \u0026amp; Fraser \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Simoes et al. 2021), as well as a semi-intensive system that involves diurnal grazing and nocturnal confinement with a regulated dietary routine suitable for their growth and reproduction compared to free-range systems (Shivakumara \u0026amp; Kiran \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Direct exposure to adverse environments could lead to heat stress occurrence. Therefore, animals managed semi-intensively are more likely to be impacted (Jack et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, THI measurements are different for both management systems and could lead to heat stress of varying severity. Therefore, this study aimed to investigate how different THI in different small ruminant management systems in a tropical environment induce heat stress and impact rams\u0026rsquo; fertility, emphasising the physiological responses and sperm quality of Barbados Blackbelly rams.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003ea)\u0026nbsp; \u0026nbsp;\u0026nbsp;Animals and Management Systems\u003c/p\u003e\n\u003cp\u003eThis study used twenty-four Barbados Blackbelly rams aged between two and three years old with body weights of between 48 and 60 kg. The animals were fed with commercial pellets and chopped Napier grass daily with \u003cem\u003ead\u003c/em\u003e\u003cem\u003elibitum\u003c/em\u003e drinking water.\u003c/p\u003e\n\u003cp\u003eThe rams were randomly divided into two groups and placed into one of two types of management system: intensive (ITS), which involved a single pen inside elevated slatted flooring pens, and semi-intensive (SIS), in which the animals could range freely outside the animal housing with no shade provided. The SIS rams were kept outside within the designated fence area, exposed to optimum sunlight with \u003cem\u003ead libitum\u003c/em\u003e water. Rams in the semi-intensive system were allowed to range for six hours daily from 9:00 am to 3:00 pm over a two-month period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eb)\u0026nbsp; \u0026nbsp;\u0026nbsp;Location\u003c/p\u003e\n\u003cp\u003eThe study was conducted at the Glami Lemi Biotechnology Research Centre Universiti Malaya, Jelebu, Negeri Sembilan, Malaysia (3° 3' 50.2452'' N; 102° 3' 26.7588'' E). The sheep barn and the weather station were about 100 metres apart.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ec)\u0026nbsp; \u0026nbsp; \u0026nbsp;Climate Data\u003c/p\u003e\n\u003cp\u003eThe study was conducted in three cycles within 2022 – 2024, between February and August. The temperature and humidity data were measured by using a Temperature-Humidity logger (Elitech GSP-6) for the intensive system \u0026nbsp;(hanging on the top of the experimental ram’s pen). Meanwhile, the Temperature-Humidity Index (THI) for the animals under semi-intensive management was evaluated using a weather station, Rainwise® MKIII. The logger and the weather station recorded the temperature and humidity every 30 minutes daily for two months. Measured data were retrieved from 9:00 am to 3:00 pm with 30-minute intervals. THI values were calculated using the formulation below:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTHIs = \u0026nbsp;3.43 + (1.058) (Ta) – 0.293 × RH \u0026nbsp;+ 0.0164 × Ta × RH + 35.7\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(Berman et al., 2016)\u003c/p\u003e\n\u003cp\u003eTHI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;= Temperature-Humidity Index\u003c/p\u003e\n\u003cp\u003eTa \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;= Ambient temperature, °C\u003c/p\u003e\n\u003cp\u003eRH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;= Relative humidity as a fraction of the unit, %\u003c/p\u003e\n\u003cp\u003ed)\u0026nbsp; \u0026nbsp;\u0026nbsp;Physiological Assessment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhysiological parameters such as rectal temperature (RT) (measured using a rectal thermometer), heart rate (HR) per minute (a CMS60D-VET pulse meter), respiration rate (RR) per minute (flank movement), and testicular temperature (TT) (an infrared thermometer) were assessed weekly after 3:00 pm. To reduce the effect of vigorous movement during the assessment procedures, the rams were kept calm for five minutes before any assessment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ee)\u0026nbsp; \u0026nbsp; \u0026nbsp;Sperm Quality\u003c/p\u003e\n\u003cp\u003eSemen was collected using an artificial vagina (AV) weekly at 3:00 pm for two months. The semen was diluted with a Tris-Citrate Acid Yolk Extender (TCAYE) before sperm kinematics and viability characteristics evaluations were conducted using a Computerised Assisted Sperm Analyser (CASA), IVOS (Hamilton Thorne). The semen was diluted 100- and 10-fold for the kinematic and viability evaluations, respectively. About 10 µL of diluted sperm was placed onto a Makler chamber, and three technical replicates were recorded for each sample. Sperm kinematic parameters from the intensive and semi-intensive systems were evaluated using IVOS (Animal motility module) version 12, with the following settings: 30 frames at a frame rate of 60 frames/s; minimum contrast = 20; minimum cell size = 5 pixels; motility = 30 μm/s; progressive motility = 50 μm/s; straightness = 70%. The Makler chamber was heated to 37 °C prior to the sample observation. The kinematic-related variables evaluated were the VAP (velocity average path), VCL (curvilinear velocity), VSL (straight-line velocity), BCF (beat cross frequency), ALH (amplitude of lateral head displacement), total and progressive motility, and percentage of cells with rapid, medium, slow, and static movements for the sperm velocity distribution.\u003c/p\u003e\n\u003cp\u003eSperm viability and abnormalities (head, midpiece, and tail) were assessed using premixed eosin-nigrosin staining (Nidacon®). A total of 200 sperm were counted with three technical replicates for each sample. The sperm quality were also measured at 1, 2, and 4 hours after incubation in a water bath at 37 °C for thermoresistance test (Lange-Consiglio et al., 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ef)\u0026nbsp; \u0026nbsp; \u0026nbsp;Hormone and oxidative stress levels\u003c/p\u003e\n\u003cp\u003eEnzyme-linked Immunosorbent Assay (ELISA) was conducted to detect testosterone, cortisol and malondialdehyde (MDA) levels using pre-coated FineTest\u003csup\u003eⓇ\u003c/sup\u003e ELISA plates. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was analysed using CheKine\u003csup\u003eTM\u0026nbsp;\u003c/sup\u003eMicro colourimetric assay. Hormone levels were evaluated using blood serum, while oxidative stress markers were evaluated using seminal plasma.\u003c/p\u003e\n\u003cp\u003eg)\u0026nbsp; \u0026nbsp; \u0026nbsp;Statistical analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData were analysed by one-way repeated measures ANOVA according to incubation time and week parameters under the different management systems and THI. Student’s t-test was used to evaluate the differences between the means of the two treatment groups. All statistical analyses were conducted at a significance level of \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u0026nbsp;or \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 using IBM SPSS software version 29. Data were expressed as means and standard error.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTemperature-Humidity Index (THI)\u003c/h2\u003e \u003cp\u003eThe THI in the SIS was significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) compared to that in the ITS. Both management systems entered the danger range of the THI at 9:00 am. The SIS reached an emergency THI range earlier than the ITS, indicating that the SIS rams were exposed to severe discomfort earlier and longer than their ITS counterparts. Despite that, the Pearson correlation test showed a positive correlation between the THI of both ITS and SIS (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.531, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05),\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhysiological responses\u003c/h3\u003e\n\u003cp\u003eThe discomfort level, as reflected in all the physiological parameters, is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The SIS rams recorded significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) HR, RR, RT, and TT compared to the ITS rams. The current study also indicated that there was no significant interaction between the weeks and management systems (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, the weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) did not influence the physiological responses in either the ITS or SIS rams (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). TT of the SIS rams at weeks 1, 4, 5, 7, and 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed no significant differences with the TT of the ITS rams, although the SIS rams had higher TT. The Pearson correlation test found the THI in the ITS was not correlated with the HR (\u003cem\u003er\u003c/em\u003e = -0.026, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), RT (\u003cem\u003er\u003c/em\u003e = -0.124, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), or TT (\u003cem\u003er\u003c/em\u003e = -0.172, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), except RR which showed a significant correlation (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.285, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Meanwhile, significant correlations were recorded between the THI and the HR (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.207, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), RT (r\u0026thinsp;=\u0026thinsp;0.302, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), TT (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.172, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and RR (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.217, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for all physiological assessments of the rams in the SIS,\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysiological parameters of rams in different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eManagement System\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeart rate (beats/minute)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e80.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e114.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRespiration rate (breaths/minute)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e86.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e135.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRectal temperature (\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e39.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTesticular temperature (\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e36.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e36.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. * indicates a significant difference in the same row using T-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSperm quality\u003c/h3\u003e\n\u003cp\u003eThe current study recorded no significant differences between the management systems\u0026rsquo; semen volume and sperm concentration of the BB rams (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The sperm viability of the ITS rams was significantly better than that of the SIS rams. Meanwhile, in relation to sperm abnormality (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the head, midpiece, and tail abnormalities of the sperm of the SIS rams were significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those of the ITS rams. This was also evident in the increased susceptibility of the sperm to abnormalities and the more rapid decline in sperm quality with prolonged incubation, compared to rams maintained under the ITS. (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSemen volume and sperm concentration of rams in different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eManagement Systems\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemen volume (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm concentration (10\u003csup\u003e9\u003c/sup\u003e/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. * indicates a significant difference in the same row using T-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSperm viability and morphology abnormalities of rams in different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eManagement Systems\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eViability, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e92.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e88.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbnormality, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHead abnormality, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMidpiece abnormality, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTail abnormality, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. * indicates a significant difference in the same row using T-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSperm viability and morphology abnormalities of rams over different incubation times and under different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIncubation Time (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eManagement systems\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eViability, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003csup\u003eb,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAbnormality, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. Superscript \u003csup\u003ea,b\u003c/sup\u003e indicates a significant difference in the same row within the same parameters using T-test \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Superscript \u003csup\u003ew,x,y,z\u003c/sup\u003e indicates a significant difference in the same column within the same management system using repeated measures ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eData presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that the sperm motility and progressive motility of the SIS rams were significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than ITS rams. The results revealed a significantly greater deterioration in sperm parameters of SIS rams compared to ITS rams after 4 hours of incubation. Other kinematic parameters indicated notable differences in the results regardless of the incubation time, with the VAP, VSL, and VCL recorded as being significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the SIS rams (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Nonetheless, no notable differences in ALH and LIN were observed between the fresh readings and those taken after 1 hour of incubation hour. Meanwhile, no significant difference was found between the management systems for STR during 1 hour of incubation. Nonetheless, for incubation times of 2 and 4 hours, the kinematic parameters exhibited consistent significant trends similar to other kinematic parameters, with the SIS rams\u0026rsquo; sperm recorded as significantly lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to their ITS counterparts for ALH, LIN, and STR.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSperm motility and progressive motility over different incubation times and under different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIncubation Time (h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eManagement systems\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTotal motility, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003eb,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eProgressive motility, (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003eb,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. Superscript \u003csup\u003ea,b\u003c/sup\u003e indicates a significant difference in the same row within the same parameters using T-testing (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Superscript \u003csup\u003ew,x,y,z\u003c/sup\u003e indicates a significant difference in the same column within the same management system using repeated measures ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinematic parameters over different incubation times and under different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIncubation Time (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eManagement systems (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePath velocity (VAP), \u0026micro;m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003e,a,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1.08\u003csup\u003e,a,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003eb,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eProgressive velocity (VSL), \u0026micro;m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003eb,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTrack speed (VCL), \u0026micro;m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e185.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e172.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e159.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e135.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e121.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003csup\u003eb,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59\u003csup\u003ea,w\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLateral amplitude (ALH), \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eStraightness (STR), %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003eb,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLinearity (LIN), %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Fresh)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003csup\u003ea,z\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003ea,y\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. Superscript \u003csup\u003ea,b\u003c/sup\u003e indicates a significant difference in the same row within the same parameters using T-testing \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Superscript \u003csup\u003ew,x,y,z\u003c/sup\u003e indicates a significant difference in the same column within the same management system using repeated measures ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e\u003cb\u003eHormones and oxidative stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMeanwhile, the average testosterone and cortisol concentration (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) showed no significant difference between ITS and SIS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), despite the testosterone concentration of ITS being slightly higher compared to SIS, indicating a trend due to the impact of the heat stress, especially for SIS., consistent with physiological and sperm quality parameters Concurrently, the cortisol concentration of SIS was slightly higher than that of ITS, despite no significant difference recorded in both systems. Oxidative stress indicators such as malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were significantly higher in SIS seminal plasma than ITS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHormones oxidative stress levels in different management systems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHormones\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSIS (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTestosterone, ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160.57\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150.69\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCortisol, ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e235.28\u0026thinsp;\u0026plusmn;\u0026thinsp;12.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e243.10\u0026thinsp;\u0026plusmn;\u0026thinsp;18.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOxidative Stress\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalondialdehyde, nmol/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydrogen Peroxide, \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e133.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. **indicates a significant difference in the same row of the same treatment using T-test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eA higher THI was recorded in the SIS than the ITS, reflecting the ram experiencing discomfort. Referring to Thom (1957) and supported by Hahn et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Berman et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the THI ranges of severity classification for ruminants are as follows: normal\u0026thinsp;\u0026le;\u0026thinsp;74; moderate 75\u0026ndash;78; severe 79\u0026ndash;83; and very severe (emergency)\u0026thinsp;\u0026ge;\u0026thinsp;84. The ITS effectively regulates the THI in the immediate environment of the animals, which is especially important during extreme weather events when THI levels may spike significantly. The THI correlation of ITS and SIS, indicating that the climate conditions to which the SIS was exposed directly also affected the THI in the ITS.\u003c/p\u003e \u003cp\u003ePhysiological responses such as HR, RR, RT, and TT have been reported as reliable indicators of heat stress occurrence in small ruminants (Sejian et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Al-Dawood 2007; Marai et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These responses are part of the thermoregulatory countermeasures to mitigate excessive heat load in the body (Moula et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ghezzi et al. 2023; Li et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The current study revealed that the SIS rams experienced greater heat stress compared to the ITS rams. A primary reason for this was the exposure of the SIS rams to solar radiation, leading to a higher radiant temperature compared to the air temperature observed in the ITS (Sch\u0026uuml;tz et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, the ITS rams were also exposed to a very severe THI range based on Hahn et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) classification. However, their physiological responses, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, did not reflect stress behaviour, in contrast to the responses of the SIS rams. Previous reports indicated the physiological responses of Santa Ines rams under heat stress, which recorded a respiratory rate (RR) of 113 breaths per minute, a heart rate (HR) of 134 beats per minute, and a rectal temperature (RT) of 39.6\u0026deg;C (Gesualdi et al. 2014; Rashamol et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, the air temperature in the ITS was assumed not to affect the rams significantly, even though the THI exceeded the emergency threshold. This suggests that the THI range of classification was not ideal for the ITS in the tropics, and the fact that minimal effects were observed may be attributed to the breed-specific characteristics of the Barbados Blackbelly sheep, which showed no significant physiological responses despite the THI threshold for heat stress being surpassed. Furthermore, the adaptive traits of Barbados Blackbelly rams to tropical adverse environmental conditions may help mitigate the effects of heat stress (Paim et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; de Almeida et al. 2018; Horton \u0026amp; Burgher \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). It was also reported by Maurya et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) that the effects of heat stress on sheep differ depending on their sex, where it has been reported that males exhibited better adaptability to heat stress than females. Females have greater susceptibility to heat stress, which is reflected in their severe physiological, endocrine and blood-biochemical responses than males (Maurya et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe correlation of RR with THI in ITS is due its efficiency for physiological response that results in a more vigorous mechanism for eliminating excessive heat load from the body through respiratory tract evaporation, ensuring a homeothermic condition (Silva et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although the THI value recorded in the ITS exceeded the threshold designated for emergency conditions, the physiological responses detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e do not correspond with the anticipated severity of heat stress. This indicates that the THI range may not have substantially impacted the ITS rams' physiological status. This could be reflected in, for example, heart rates of 80 beats/minute in a resting state (Yaxley et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and 90\u0026ndash;95 beats/minute in a normal state (Shilja et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which are equivalent with the data obtained for the ITS rams. This indicates that the ITS rams were not experiencing extreme heat stress, suggesting that the existing common THI ranges for heat stress severity may not be suitable for BB sheep in the tropics. According to Berman et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the common THI classifications were mostly based on ambient temperature (Ta). This overemphasised the temperature diminished the significance of humidity in the animal heat stress, particularly in tropical regions, where humidity is more prominent. Because the THI index might not adequately reflect the combined effects of temperature and humidity, the animals might not show physiological stress reactions even though the THI readings fall into the emergency category (\u0026ge;\u0026thinsp;84). This is especially important in settings where humidity contributes significantly to heat stress but is not sufficiently considered when calculating THI.\u003c/p\u003e \u003cp\u003eThe significant correlation of SIS for all physiological parameters with the THI implying that they were exposed to more heat stress as the THI rose. The correlations suggest that the SIS rams displayed significant physiological responses to maintain normothermia conditions and regulate their body temperature when approaching the upper limit of their thermoneutral zone (P\u0026eacute;rez et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sch\u0026uuml;tz et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings align with those of a previous study by Mascarenhas et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who reported a correlation between physiological responses and THI. Furthermore, in this study, TT was consistently lower than the RR for the entire research period. This implied that testicular thermoregulation had occurred to maintain a TT below the body core temperature. Increased TT due to heat stress can have detrimental effects on the seminiferous tubule diameter (STD) and testicular weight, which could subsequently lead to detrimental effects on the epididymal sperm and spermatids (Teixeira et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Severe heat stress can impair testicular function by inducing oxidative stress, thereby disrupting intracellular signal transduction (Lekola et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ahsan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe spermatogenesis cycle in rams lasts 49 days, leading to the storage of sperm in the epididymis prior to ejaculation. It has been reported that the deleterious effect of heat stress will not affect mature spermatozoa as its effect occurs mainly during earlier stages of sperm development (Sierra et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rizzoto \u0026amp; Kastelic \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; El Amiri 2020). Thus, minimal changes were observed in the semen volume and sperm concentration under both management systems. Nevertheless, heat stress has been discovered to negatively influence sperm concentration in dorper rams during summer, indicating a period of high ambient temperature (Barragan et al. 2023). Based on the current study, most of the recorded sperm quality parameters indicated more negative impacts on the SIS rams compared to the ITS rams. Sperm morphology is impaired during heat stress due to detrimental changes in the meiosis cycle of the spermatocytes and spermiogenesis. Prolonged exposure to chronic heat stress exceeding two months could result in 3.6% of sperm developing major abnormalities and 43.4% developing minor abnormalities (Moula et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Meanwhile, short exposure to heat stress could also lead to more sperm abnormalities due to sperm epididymal damage (Barragan et al. 2024). The effect of sperm morphology impairment after a brief exposure to heat stress may be observed after several weeks or as early as two weeks after the exposure, and the effects might persist for a minimum of eight days and last for up to 77 days (Shahat et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Abdelhamid et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Consequently, the effects may persist up to two months, although the exposure to heat stress has been diminished (Shahat et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The data in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e also align with previous studies on boar sperm. The previous study reported that exposure to heat stress resulted in an increase in sperm abnormalities, with no effects on the semen volume or sperm concentration (Rizzota \u0026amp; Kastelic 2020; Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cameron \u0026amp; Blackshaw,1980).\u003c/p\u003e \u003cp\u003eThe sperm motility and progressive motility deteriorated severely after four hours of incubation, implying that exposure to heat stress under the SIS could increase sperm susceptibility. In addition, repeated measures analysis showed significant interactions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between the management system and the incubation time. This implies that the management system and incubation time both affect the total motility and progressive motility of the sperm. These new findings are also supported by previous studies and reviews by Barrag\u0026aacute;n et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Moula et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who reported a major decrease in the sperm quality of rams under heat stress. Meanwhile, in line with other kinematic parameters, the ITS rams exhibited significantly more rapid sperm than the SIS rams. It is critical to maintain sperm with rapid velocity as these have greater potential to penetrate the cervical mucus, increasing the potential for fertilisation, while slower sperm may hamper fertilisation (Johnson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ford et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe decrease in sperm thermoresistance in rams reared in the SIS could be attributed to the build-up of reactive oxygen species (ROS), leading to oxidative stress and lipid peroxidation during heat stress exposure. This cascade chemical reaction negatively affects sperm motility (Teymoori et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Qamar et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dutta et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, the downregulated of several genes related to the translation of carboxypeptidase Q-precursor (\u003cem\u003eCPQ\u003c/em\u003e), superoxide dismutase (\u003cem\u003eSOD1\u003c/em\u003e) and NPC intracellular cholesterol transporter 2 (\u003cem\u003eNPC2\u003c/em\u003e) that are responsible in activation of hormones peptides and oxidative stress regulation as well as epidydimal transit functions were also reported to occur concurrently as the sperm motility in rams deteriorated during heat stress (Neto et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the SIS rams were more affected by heat stress than ITS, which was also supported by physiological response results shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, where the RT, TT, HR and RR were significantly higher in SIS rams. The optimal kinematic parameters must be attained as these are responsible for determining animal fertility, especially VAP, which is highly correlated with animal fertility status (Tanga et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nagy et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, the current study indicates that sperm from an SIS may result in reduced ram fertility compared to sperm from an ITS. At first, both the STR and LIN parameters in the management systems decreased for an hour before starting to recover after 2 hours of incubation. The recovery observed could be attributed to the spermatozoa\u0026rsquo;s adaptability to the extender microenvironment, which has been reported to affect sperm functionality (Neila-Montero et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Meanwhile, the VCL and STR were significantly higher in the ITS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the SIS. Higher VCL and STR are known to reflect higher sperm hyperactivation; this is crucial for zona pellucida penetration, which is a prerequisite in fertilisation (Suarez \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Furthermore, the VCL and VAP of the sperm have been reported to play critical roles in fertilisation due to their major attribute of transporting spermatozoa through the mucus of the ewe cervix (Van de Hoek et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, it is known that testosterone plays a critical role in maintaining spermatogenesis (Sharpe et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The fact that the current study showed deteriorated sperm quality, especially for SIS, indicates an adverse effect of testosterone concentration during the early sperm maturation process. The elevated cortisol levels represent a component of the heat stress response observed in the SIS rams, which was triggered by the hypothalamic-pituitary-adrenal (HPA) axis and the sympatho-adrenal-medullary (SAM) system. This response leads to adrenocorticotropic hormone (ACTH) secretion, subsequently stimulating glucocorticoid production, including cortisol (Barrag\u0026aacute;n Sierra et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It could be interpreted that an increase in reactive oxygen species (ROS) due to heat stress in SIS led to oxidative damage through multiple pathways, such as the peroxidation of polyunsaturated fatty acids (PUFAs) affect the sperm membranes, DNA fragmentation, and impaired mitochondrial function, where the impacts collectively deteriorate sperm quality and ultimately affecting fertilisation capability. Moreover, heat stress amplifies ROS production and reduces antioxidant defences in seminal plasma, creating a synergistic effect that heightens the harmful influence of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) on sperm quality, leading to a decline in fertility potential (Agarwal et al. 2014; Durairajanayagam et al. 2015). The H₂O₂ impact includes irreversible axoneme damage responsible for protein production in the flagellum (Aitken et al. 2012). Despite having a higher MDA concentration than in ITS, the concentration of MDA in SIS was less than 3 nmol/L, indicating the MDA concentration was still within the fertile range based on Almady et al. (2021). The other parameters were also aligned with the MDA data, indicating that most of the SIS parameters were documented as being more severely affected by heat stress than those of the ITS. Still, it is noteworthy that the severity remained within an acceptable range and did not lead to infertility.\u003c/p\u003e \u003cp\u003eIn conclusion, exposing Barbados Blackbelly rams to a SIS reduces the effects of heat stress on their physiological, reproductive, and oxidative stress levels compared to an ITS. However, the impact does not extend to infertility in the rams. The negative effects on reproductive performance stay within an acceptable range. Thus, the semi-intensive system may remain in use, provided that effective mitigation measures are implemented to minimise the effects of heat stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eThe use of animals in the study was approved by the Universiti Malaya Institutional Animal Care and Use Committee (UM IACUC) with reference number \u003cb\u003eS/28062022/03062022-01/R.\u003c/b\u003e\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest statement:\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor ORCID\u003c/h2\u003e \u003cp\u003eMohd Shahmi Hakimi Mazlishah \u0026minus;\u0026thinsp;0000-0003-0221-3600\u003c/p\u003e \u003cp\u003eNuradilla Mohamad-Fauzi \u0026minus;\u0026thinsp;0000-0001-5192-9889\u003c/p\u003e \u003cp\u003eMohd Fadzil Firdzaus Mohd Nor \u0026minus;\u0026thinsp;0000-0002-6145-6381\u003c/p\u003e \u003cp\u003eMuhammad Syafiq Roslan \u0026minus;\u0026thinsp;0009-0007-7370-9247\u003c/p\u003e \u003cp\u003eShamsul Azlin Ahmad Shamsuddin \u0026minus;\u0026thinsp;0000-0001-7983-6502\u003c/p\u003e \u003cp\u003eNoor Hashida Hashim \u0026minus;\u0026thinsp;0000-0003-2572-4083\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was funded by the Ministry of Higher Education (MOHE) under the Fundamental Research Grant Scheme (FRGS/1/2022/STG03/UM/02/7).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eMazlishah, M.S.H., wrote the first draft and carried out the experiment, data collection, optimisation, and correction. Hashim, N.H., reviewed the draft, supervised the study, and made the experimental design. Mohamad-Fauzi, N. reviewed the draft, proofread it, and the source of funding. Nor, M.Z.M. reviewed the draft and source for the climate data referral expert. Shamsuddin, S.A.A. optimised computerised assisted sperm analyser parameters. Roslan, M.S., assisted in the study and data collection in the field.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data used in the study are available from the corresponding author, Hashim, N.H upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelhamid MHM, Walschaerts M, Ahmad G, Mieusset R, Bujan L, Hamdi S (2019) Mild experimental increase in testis and epididymis temperature in men: effects on sperm morphology according to spermatogenesis stages. Transl Androl Urol 8(6):651.\u003c/li\u003e\n\u003cli\u003eAgarwal A, Parekh N, Selvam MKP, Henkel R, Shah R, Homa ST ... 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Sci Rep 11(1):7803.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heat stress, reproductive performance, sperm quality, hormones, oxidative stress, physiological responses","lastPublishedDoi":"10.21203/rs.3.rs-6779838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6779838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTropical regions face climate change, which increases the impacts of heat stress (HS) on ram fertility. Twenty-four Barbados Blackbelly rams were divided into intensive (ITS) and semi-intensive (SIS) management systems. ITS rams were housed in a barn, while SIS rams spent six hours daily outside over two months. Daily climate data were collected, and the Temperature-Humidity Index (THI) was calculated. Weekly semen collections and physiological assessments were conducted. Sperm motility was evaluated using a Computerised Assisted Sperm Analyser (CASA), alongside sperm viability, morphology, hormone levels, and oxidative stress. SIS rams experienced more severe heat stress, indicated by higher physiological responses (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than ITS rams. SIS sperm quality was significantly lower, with reduced motility, progressive motility, kinematic parameters, morphology, and viability (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Testosterone and cortisol levels showed no significant differences between systems, though ITS had higher testosterone and SIS had elevated cortisol. MDA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) followed similar trends, with SIS rams showing higher oxidative stress levels. The THI for SIS was significantly greater than that of ITS, potentially affecting adaptable breeds like Barbados Blackbelly, resulting in lower fertility in SIS rams. Despite exceeding the THI threshold, the minimal impact on physiological responses in ITS rams suggests a contradiction, indicating a need for a specific classification of heat stress severity, with a focus on humidity in the tropics. Although heat stress (HS) impacts reproductive performance, physiological functions, and oxidative stress parameters, it does not significantly lead to infertility, and the rams generally remain fertile.\u003c/p\u003e","manuscriptTitle":"Heat stress impact on reproductive and physiological performance of Barbados Blackbelly rams under different management systems in the tropics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 10:55:19","doi":"10.21203/rs.3.rs-6779838/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d255f7a-8816-4a11-9899-d5fd6641aec2","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-24T09:33:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 10:55:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6779838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6779838","identity":"rs-6779838","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}