Selenium efficiency in preserving sperm quality and testicular characteristics of South African indigenous Zulu Rams exposed to heat stress

doi:10.21203/rs.3.rs-3956893/v1

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Selenium efficiency in preserving sperm quality and testicular characteristics of South African indigenous Zulu Rams exposed to heat stress

https://doi.org/10.21203/rs.3.rs-3956893/v1

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published 19 Oct, 2024

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The purpose of the study was to investigate selenium efficiency in preserving sperm quality and testicular parameters of the hardy South African indigenous Zulu Rams exposed to heat stress. A total of 20 indigenous Zulu rams between the ages of 2–5 years were used for semen collection. The rams were allocated into 4 groups namely: Selenium (Se); Selenium and testicular heat stress (SeTHs); Testicular heat stress (THs) and Control with Each group having five rams. The groups were balanced according to body weight and scrotal circumference. Selenium and SeTHs groups received sodium selenite orally on a bi-weekly basis for a period of 4 months. To induce heat stress, testicular heat insulation bags were wrapped around the testes of the testicular heat insulated (THs) ram group for 49 days to cover the duration of spermatogenesis. Semen was collected weekly from all 20 rams for 3 months while the 1st month was for selenium acclimatization, and testicular measurements were taken bi-weekly. ANOVA was used to separate mean values for sperm quality and Duncan was utilised to compare significant differences using Statistical Analysis Software. It was observed that selenium, SeTHs, and control group obtained similar total sperm motility, while THs group recorded a low (p < 0.05) total sperm motility. SeTHs group had a significantly high (p < 0.05) medium moving (92 ± 3.5) sperm cells as opposed to the other treatment groups. The semen collected from the THs group of rams displayed a significantly higher (p < 0.05) number of immotile sperm cells. The scrotal circumference for the rams supplemented with selenium was smaller (p < 0.05). The current study concluded that induced heat stress compromised the sperm quality parameters such as sperm motility, progressive motility and kinematic parameters. Furthermore, Se supplementation protected sperm cells against testicular heat stress whereby similar sperm quality results were observed in the semen from the group of rams supplemented with selenium including those that received selenium and were exposed to testicular heat insulation.

Small stock (sheep & goats) plays a vital role in the global food and wool production in tropical and subtropical areas which are greatly affected by harsh climatic conditions (Ngcobo et al., 2022). Sheep are a major source of revenue and the backbone of many South African rural communities. There wouldn't be enough food in many rural regions without the revenue from their products. (Cloete et al., 2014). The South African Indigenous sheep breeds (including the Damara, Namaqua Afrikaner, Pedi and Zulu) are adapted to the South African arid environmental conditions (Molotsi et al., 2019). These indigenous sheep play a vital role in the livelihoods of people as a source of meat, milk, wool hide and manure (Kunene et al., 2014; Selepe, 2018). Amongst these indigenous breeds, the Zulu sheep is indigenous to the Kwa-Zulu Natal province of South Africa. The sheep breed is renowned for its adaptability, durability, and cheap upkeep. (Fossey et al., 2007). However, the genetic potential and productivity of Zulu sheep is deteriorating due to indiscriminate type of breeding (Scholtz, 2005). In conservation strategies, the ram is an important member of the flock as it contributes 50% towards the genetic potential of the flock (Serrano et al., 2021). Mainly, the ram's sexual behavior and semen quality are what restrict its ability to procreate. (Zaher et al., 2020). Environmental elements like humidity, daylight hours, and ambient temperature have an impact on the quality of semen. (Kabukçu et al., 2020). Changes in temperature and humidity might interfere with spermatogenesis and induce thermal discomfort. (Zaher et al., 2020). Climate conditions in South Africa's subtropical regions are harsh throughout the summer, which can lower male fertility by affecting the quality of semen (Llamas-Luceño et al., 2020). Heat stress caused by high ambient temperatures supresses testicular functions through oxidative damage by producing reacting oxygen species (ROS) and further changing the intracellular signal transduction (Ahsan et al., 2014). Spermatozoa are typically shielded from oxidative damage by antioxidants and antioxidant enzymes found in the seminal plasma. (Shaliutina-Kolešová et al., 2018). Spermatozoa are especially vulnerable to oxidative stress because they have poor intracellular antioxidant activity, which may not be sufficient when ROS levels surpass normal values. (Martín-Hidalgo et al., 2020). Much attention has been focused on selenium since it is an important trace element, possesses antioxidant qualities that are vital for embryonic growth and reproduction, and can successfully repair cell damage caused by stress. (Petrujkić et al., 2014). The testis requires selenium for the production of testosterone and for the growth and development of spermatozoa (Petrujkić, 2014; Cannarella, 2021). Selenium is highly required during spermatogenesis and semen collection to fight oxidative stress (Ebeid, 2009). Normally, animals obtain selenium from the grass or feed they eat (Hemingway, 2003), but in the Kwa-Zulu-natal area of South Africa, heavy rainfall has left the soil and plants lacking in the mineral. (Shabalala et al., 2013). Therefore, the goal of the current investigation was to determine how well selenium preserved the testicular parameters and sperm quality of resilient indigenous Zulu Rams from South Africa who were subjected to heat stress.

Study site and management of animals

The study was conducted at the University of Zululand Dairy farm and further analysis were done at the University’s Department of Agriculture laboratory., KwaZulu-Natal province, South Africa. The rams were separated from the ewes for the duration of data collection. The rams fed and allowed to graze on natural pastures and supplemented with lucerne. Water was provided at libitum.

Experimental design

The experiment was conducted from summer to Autumn (January-May). A total of twenty (n = 20) indigenous Zulu rams between the ages of 2–5 years were used for the study. The rams were grouped according to body weight and scrotal circumference (Table 1) and allocated to four treatment groups with each group having a total of 5 rams. The treatment groups were arranged as follows: In the first group, the rams (n = 5) were given Selenium (Se) only; in the second group, the rams (n = 5) were only subjected to testicular induced Heat stress (THs); in the 3rd group the rams (n = 5) were subjected to the combination of Selenium and testicular induced heat stress (SeTHs); lastly in forth group the rams (n = 5) were not treated with neither dietary selenium nor heat stress, this served as the control group. The rams subjected to dietary selenium were given sodium selenite orally at a dosage of 0.34 Se mg/kg of body weight. Dietary selenium was administered once fortnightly for a period of 4 months. Heat stress was induced by wrapping a thermal testicular insulation bag around the scrotum of the rams. The rams only wore testicular insulation bag for a period of 49 days, however in those 49 day the rams wore the bags for one week and rested for the next week. The 49 days covered the first spermatogenesis, the rams did not wear the insulation bags for the 4 months of data collection in order to avoid permanent damage to the sertoli cells and the scrotal tissue.

Table 1

Body weight and scrotal circumference of the experimental groups:
	Group 1 (Se)	Group 2 (SeTHs)	Group 2 (THs)	Group 4 (Control)	P-value
Body weight	39,7	39,6	38,26	40,56	0.92
Scrotal circumference	29,16	29	28,5	29,6	0.93

Semen Collection and procedures

Semen was collected by means of an electro ejaculator once a week for a period of 5 months. Scrotal circumference and testicular parameters were assessed bi-weekly using a tape measure for scrotal circumference and a vernier calibre for testicular parameters. The measurements taken for testicular parameters were classified as testicular length for right (TL Right) and left (TL Left); and testicular breath for right (TB Right) and left (TB Left). Immediately after semen collection, semen was transported to the lab for macro (volume, appearance, pH) and micro evaluations (Motility, concentration, viability).

Sperm quality analysis by CASA

Sperm quality parameters were evaluated using a computer assisted sperm analysis (CASA) system of sperm class analyser (SCA). Briefly 10 µl of semen was diluted in 200 µl of saline in an Eppendorf tube at 37°C for swim up. A 10 µl drop of the diluted semen was gently pipetted on a glass slide covered with a cover slip and placed under the microscope. Observations were done at X10 magnification using motility/concentration system of the SCA. The sperm progression was classified as total sperm motility (TSM), progressive motility (PM), none progressive motility (nPM) and immotile (IM). Sperm velocity was classified as rapid (RV) medium (MV) and Slow (SV). The sperm kinematic parameters were classified as curve speed (VCL), linear speed (VSL) straight index (STR) and oscillation index (WOB).

Data analysis

All data collected for testicular parameters, and sperm quality parameters were analysed using the general linear model of Statistical Analysis Software (SAS). The sperm motility parameters (TSM, PM, NPM and IM), Kinematics (VCL, VSL, STR and WOB) and testicular parameters were analysed as repeated measures, considering binomial distribution using Prog GLIMMIX procedure. Analysis of variance was used to separate the means. The Duncan test was used to determine the mean differences and were considered significant when (p < 0.05). Data is presented as Mean ± SEM.

Table 2 represents Mean ± SEM of sperm quality parameters of indigenous Zulu rams following selenium supplementation and induced testicular heat stress. A significantly low total Motility was observed (Table 2) when heat stress was compared with the rest of the treatment groups (P < 0.05). The control group recorded a significantly high (p < 0.05) progressive motility compared to the rest of the treatment groups. A significantly high (p < 0.05) immotile sperm cells were observed for induced testicular heat stress (THs). The progressive motility for Se and SeTHS was similar (p > 0.05). However, SeTHs recorded a significantly high (p < 0.05) medium velocity as opposed to Selenium alone. A significant difference (p < 0.05) in linear speed was observed when control was compared with the rest of the treatment groups. There were no significant differences (p > 0.05) observed in kinematics parameters when Se was compared with SeTHS. Table 3 outlines the Mean ± SEM of scrotal circumference and testicular characteristics of Zulu rams that were supplemented with selenium and subjected to testicular heat stress. The significant difference (p < 0.05) was observed when scrotal circumference for selenium treated group was compared with the combination (SeTHs). There was no significant difference (p > 0.05) observed for all the testicular parameters across all the treatment groups.

Table 2

Mean ± SEM values for sperm quality parameters following heat stress and selenium supplementation
Treatments	Se	SeTHs	THs	Control
Total motility	69.4 ± 4.7^a	69.3 ± 6.7^a	55.2 ± 6.7^c	72.6 ± 4.5^a
Progressive	46.2 ± 4,14^b	46.6 ± 4.14^b	38.7 ± 4.14^c	53.3 ± 4.11^a
Immotile	31.2 ± 4.2b^a	33.1 ± 4.2^ba	44.8 ± 4.2^a	27.1 ± 4.1^b
Rapid	32.0 ± 3.7^b	14.6 ± 3.7^c	29.9 ± 3.7^b	39.7 ± 3.7^a
Medium	23 ± 3.8^b	92 ± 3.5^a	14 ± 3.6^c	21 ± 3.9^b
Slow	14.11 ± 1.4	12.97 ± 1.4	10.87 ± 1.4	12.29 ± 1.4
Curve speed	130.7 ± .5.8^b	135.9 ± 5.8^b	116.5 ± 5.8^c	139.8 ± 5.8^a
Linear speed	70.5 ± 5.2^b	74.1 ± 5.1^b	64.2 ± 5.1^c	79.5 ± 5.1^a
Straightness	67.5 ± 3.7	66.9 ± 3.2	64.3 ± 3.2	65.8 ± 3.2
Oscillation index	63 ± 2.7	66.7 ± 2.7	63.8 ± 2.7	67.2 ± 2.6

Selenium (Se); Selenium plus testicular heat stress (SeTHs); Testicular heat stress (THs); Standard error of the mean (SEM). Values with different superscripts within a row differ significantly (P < 0.05)

Table 3

Mean ± SEM values for scrotal and testicular measurement of Zulu rams
Treatments	Scrotal & testicular parameters
Treatments	SC	TL left	TL right	TB left	TB right
Se	27.94 ± 0.40^a	11.068 ± 0.21	11.03 ± 0.25	5.44 ± 0.13	5.35 ± 0.11
SeTHs	29.59 ± 0.47	11.620 ± 0.17	11.53 ± 0.15	5.44 ± 0.12	5.54 ± 0.12
THs	29.98 ± 0.28	11.52 ± 0.19	11.75 ± 0.20	5.59 ± 0.12	5.56 ± 0.12
Control	29.17 ± 0.48	11.34 ± 0.18	11.27 ± 0.23	5.67 ± 0.13	5.67 ± 0.14

Selenium (Se); Selenium plus testicular heat stress (SeTHs); testicular heat stress (THs); Scrotal circumference; Testicular length left (TL left); Testicular length right (TL right); Testicular breath left (TB left); Testicular breath right (TB right); Standard error of the mean (SEM)

The presence of adequate selenium in the male reproductive system is paramount for normal spermatogenesis and it has a fundamental role in sperm maturation and thereby increases chances of conception (Ahsan et al., 2014). In the current study, it was shown that the control group's total sperm motility for rams was comparable to the group that received a selenium supplement. This was not in agreement with the study of Salas-Huetos et al. (2018) who conducted a meta-analysis study which discovered that selenium improved sperm motility and thereby increased chances of conception. The sperm quality parameters of the rams exposed to testicular heat stress without selenium supplementation (THs) were impaired, including total sperm motility and fast moving sperm cells, as well as sperm kinematics with curve speed and linear progression, similarly as observed by (Hosny et al., 2020) whereby sperm quality of rabbit bucks was compromised by natural heat stress. Moreover, high testicular temperature influences the anatomy of the seminiferous epithelium, testis echotexture, and pituitary and gonad hormone secretion. (Rocha et al., 2015). Selenium did shield spermatozoa from heat stress, as evidenced by the nonsignificant differences in total sperm motility, progressive motility, nonprogressive motility, immotile sperms, rapid and slow sperms for Se and SeTHS. As a result, the consumption of selenium increased the activity of the antioxidant glutathione peroxidase, which in turn increased fertility and shielded sperm cells from heat stress. (Qazi et al., 2019). Similarly, to the study of Ebeid, (2009), According to the study's findings, dietary selenium decreased lipid peroxidation and shielded sperm from free radical damage, which improved sperm motility overall and decreased the proportion of immotile sperm under settings where testicles were heated. When compared to the other treatment groups, the control group exhibited high progressive motility. Even though the testicular heat stress group supplemented with selenium displaced a satisfactory overall motility, the majority of the sperm cells in that group were surprisingly medium moving sperm cells. The only variable that varied between the two groups receiving selenium supplementation was this medium velocity (Se & SeTHs). The overproduction of reactive oxygen species and the animal's exposure to heat stress are blamed for the detrimental effects, which include changes in testicular structure and weight as well as lower-quality sperm (Hosny et al., 2020). There were a lot of immotile sperm cells in the semen collected from the rams that had been subjected to testicular heat stress, indicating that heat stress had a detrimental effect on sperm quality. The study observed that induced heat stress also comprised the Kinematic parameters as observed in the semen of the testicular induced heat stress group of rams. However, comparable kinematic values were noted for testicular heat stress and selenium, demonstrating the effectiveness of selenium in maintaining sperm quality parameters. The sperm cells' linear speed was impacted by testicular heat stress. It's interesting to note that the group receiving selenium supplements seems to have a reduced scrotal diameter., which disagrees with the results presented in the study of Qazi et al., (2019), whereby selenium supplementation increased testicular size of male animals. The effects of heat stress or selenium supplementation on testicular breath were not seen. In comparison to selenium, the control group showed better overall sperm quality measures (total motility, progressive motility), and some parameters (immotile sperm cells, rapid velocity, medium velocity) were similar for both groups. This may be explained by the fact that the organic isotopes of selenium are absorbed more quickly than the inorganic isotopes, and by the intestine's poor retention of selenite relative to organic isotopes like selenomethionine (Ha et al., 2019). According to the study, sperm quality indicators like kinematics, progressive motility, and sperm motility were all negatively impacted by heat stress. The results showed that sperm quality metrics were similar in the selenium (se) and selenium plus testicular heat stress (SeTHs) groups, suggesting that selenium supplementation protected sperm cells against testicular heat stress. Based on the study's findings, we also came to the conclusion that because indigenous Zulu sheep in South Africa are already acclimated to their surroundings, selenium supplementation should only be given when temperatures rise above the threshold.

Ethics approval

The ethical clearance for this current study was granted by the University of Zululand ethics committee (UZREC 1171110-030 PGD 2021/20).

The research followed the ethical rules and policies outlined by the University of Zululand and experiments were performed according to the ethical committee’s satisfaction. Animals were treated as humane as possible according to the Animal Protection Act 71 of 1962 for the duration of data collection.

Acknowledgements

The authors would like to thank the University of Zululand research farm workers and animal science reproduction physiology honours students for their assistance with data collection. Dr F Thabethe and Prof P Njuho for statistical analysis.

Authors contributions

Lekola KPM designed the experiments; Lekola KPM & Ngcobo MS performed the experiments; Lekola KPM, Ngcobo MS & Lehloenya KC interpreted the data; Lekola KPM drafted the Manuscript; Lekola KPM, Ngcobo MS & Lehloenya KC were all responsible for reviewing and editing; Lehloenya KC Supervised the study.

Funding

This work was supported by the National Research Foundation- Thuthuka grants (TTK210409593326) and New Generation of Academic Program (nGAP).

Conflict of interest

The authors declare no competing interests.

Data availability

Data will be made available on request.

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Journal Publication

published 19 Oct, 2024

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Editorial decision: Major revision with re-assessment
07 Jun, 2024
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You are reading this latest preprint version

Selenium efficiency in preserving sperm quality and testicular characteristics of South African indigenous Zulu Rams exposed to heat stress

Status:

Journal Publication

Version 1

Abstract

Introduction

Materials and Methods

Study site and management of animals

Experimental design

Semen Collection and procedures

Sperm quality analysis by CASA

Data analysis

Results

Discussion

Declarations

References

Status:

Journal Publication

Version 1