AAVS_13_s1_111-119

INTRODUCTION

Climatic conditions can significantly affect semen quality. Indeed, sexual behavior, semen quality, and quantity are key factors limiting male reproductive efficiency throughout the year. These factors could vary according to different environmental and physiological factors such as climate (Hu et al., 2020); latitude, and breeding season of the year (Saleh and Abdul-Ameer, 2019). Semen quality can be influenced by factors such as breed, age, nutritional status, and environmental conditions, including ambient temperature, humidity, and daylight hours (Saeed and Zaid, 2019; Goshme et al., 2020). Selection of young rams for fertility can be accomplished through selecting some important reproductive traits such as age at puberty, body condition score, body growth rate, scrotal circumference, scrotal growth rate, and semen quality (Zaher et al., 2020; Maquivar et al., 2021). In general, the sexual development of rams appears to be more closely related to body growth than to their age (Andreeva et al., 2020).

The age of onset of puberty in ram lambs varies across breeds and climatic conditions (Maquivar et al., 2021). However, it is distinguished by a sequence of endocrine processes which activate the reproductive axis, making the Sertoli and Leydig cells susceptible to the effects of gonadotropins (FSH and LH), thereby initiating spermatogenesis. The photoperiod influences the timing of puberty in ram lambs, with longer daylight periods generally promoting earlier sexual maturation. Typically, puberty occurs between 20 and 28 weeks of age, when the animal has attained approximately 65% of its mature body weight (Li et al., 2024).

The anterior pituitary gland produces the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In males, FSH primarily targets Sertoli cells in the seminiferous tubules to support spermatogenesis, while LH stimulates testosterone production in the Leydig (interstitial) cells (Banihani 2020; Duan et al., 2021). The FSH hormone consists of α and β-subunits; β-subunits play a role in determining the specificity of bonds with receptors (FSHR) (Ulloa-Aguirre et al., 2018; Obaid et al., 2022). Spermatogenesis is a highly regulated and continuous process that takes place in the seminiferous tubules by which normal diploid cells transform to sperm (haploid). Spermatogenesis depends on stem cells, spermatogonia (Madeja et al., 2021).

In males, FSH promotes spermatogenesis, the process of sperm development, in the seminiferous tubules primarily via actions on Sertoli cells (Rougier et al., 2019; Barquero et al., 2021). For this reason, polymorphisms within the FSHβ gene and its promoter are of particular interest to male fertility. FSH activates the proliferation of Sertoli cells during fetal and neonatal development as well as during the pubertal phase. Since each Sertoli cell can only support a limited number of developing male germ cells at a time, it is important that we understand how polymorphisms of the FSHβ subunit gene can affect male fertility (Recchia et al., 2021).

Sperm motility is the most widely tested semen quality trait, which requires the use of an intact plasma membrane and a certain chemical gradient of ions and other soluble components to operate correctly (Jabulani et al., 2020). An unstained semen sample may be subjected to a manual microscopic examination to determine the percentage of total and/or increasingly motile sperm (Touré et al., 2021; Johannisson et al., 2024). It is a simple and fast approach that does not require costly equipment. Photographic analysis or computer assisted semen analysis (CASA) can be used to analyze sperm concentration, motility, motion, and to some degree sperm head morphology as an alternative to subjective visual motility evaluation (Tanga et al., 2021).

Sperm morphology is usually determined by spermatogenesis (Johannisson et al., 2024) or events that occur following spermiation, and improper handling techniques or problems during cooling and freezing, which can often harm the acrosome or cause sperm tail reflection (Ngcobo et al., 2020; Savvulidi et al., 2021). The objectives of this study are to provide a basis for identifying and selecting rams with superior reproductive performance and desirable genetic traits, and to evaluate the genetic potential of local Iraqi rams by examining their semen characteristics. Selection of breeds based on genetic traits is not currently practiced in Iraq, which supports future breeding programs.

MATERIALS AND METHODS

Experimental design

Samples were collected from Al-Shoáalla slaughterhouse positioned in western Baghdad as follows: A total of 50 testicular samples were collected for each age group and distributed across genotypes as follows: (AA n=16, AG n=17, GG n=17) in 1-year-old, >1-year-old, and 2-year-old rams, respectively; age is determined as possible according to the dental figures (Miller and Robertson, 1975) and information from the owners. The samples belong to local rams aged nearly (12-24) months as determined by dental formula (Albrkaty, 2024). Blood samples were collected from the jugular vein before slaughtering the animals for estimation molecular study for molecular analysis of the FSH-beta subunit gene. The epididymis was washed in tap water to remove blood and any other debris. Each caudal of epididymis was injected with 2mL of the diluent Botu-TurboTM (semen extender) warmed at 37°C, the spermatozoa were collected by the method of slicing: the visceral layer of the tunica vaginalis covering the cauda epididymis (CE) was carefully removed to prevent any blood contamination. The exposed CE was then rinsed with phosphate-buffered saline (PBS), excised, and finely sliced using a sterile scalpel in a Petri dish containing 3 mL of semen extender. The sliced tissue was left immersed in the extender in water bath at 37-38 °C to facilitate spermatozoa release. Subsequently, the suspension was gently rinsed with an additional 2 mL of semen extender and filtered through sterile gauze sheets to obtain a clear sperm sample and evaluate the physical properties of epididymal fluid, including individual motility %, dead sperms %, and abnormal sperms %.

Collection of blood before the slaughter for evaluation of a genetic study; DNA extraction of blood, PCR by using primers (Table 1), and Sequencing or Alleles by PCR-RFLP. Collection of blood samples: Investigation of the FSH-beta subunit gene. Blood Genomic DNA was extracted from the collected blood samples using the Favor Prep Blood genomic DNA extraction Mini kit.

Table 1: Primers applied for analysis of FSHβ-gene.

Gene	Sequence 5′ - 3′		Amplicon size
FSHβ	F	CTT CCA GAC TAC TGT AAC TCA TC	313bp
	R	GTA GGC AGT CAAA GC ATC CG

DNA extraction from blood

According to the manufacturer’s instructions, the protocol designated for extraction of genomic DNA from whole blood, serum, and plasma samples was applied to extract of DNA from the frozen EDTA-blood samples of the study rams.

PCR amplification

The amplified fragments represented the desired product of FSHB-subunit gene with a molecular weight of 313 bp (Figures 1 and 2).

Primer preparation

To prepare the working primer solution from stock primer, a total of 250 µl nuclease-free water was added to each tube of primers (forward and reverse) (Table 1), mixed well by vortex, and saved deep frozen.

Mastermix preparation

PCR is a technique that selectively amplifies many copies of a particular segment of DNA using a specific enzyme, polymerase, and a specific primer (s) (Joshi et al., 2010). According to manufacturer instructions, AccuPower PCR-PreMix kit (Biotechnology, Korea) was employed in the present study to prepare of MasterMix. The tubes of PreMix have a ready-to-use PCR reagent that is optimized

for more accurate amplification, as they are characterized by speed, stability, reproducibility, and simplicity. According to manufacturer instructions, the MasterMix of each sample was performed at a final volume of 25μl. Then, the tubes of MsterMix were mixed by Exispin vortex (Digsystem, Germany) at 3000 rpm for 3 minutes and transferred to the thermal Cycler. PCR primers were designed based on a previously performed reference (Yang et al., 2011) and provided by Macrogen Company, Korea. Primers were applied for analysis of the FSHB-Subunit gene as demonstrated in Table 1.

Statistical analysis

Data were analyzed using one-way analysis of variance (ANOVA) to evaluate statistically significant differences among the experimental groups. Data were organized and tabulated using Microsoft Excel, and statistical analyses were performed using IBM SPSS Statistics for Windows (version 26.0; IBM Corp., Armonk, NY, USA) following the procedures described by George and Mallery (2019). Differences among means were considered statistically significant at P < 0.05.

RESULTS

Sperm properties and genotypes of FSHβ subunits in ram of 1-year age

According to the study’s age groups, the findings, as revealed in Table 2, showed a significant association (P<0.05) between sperm properties and the studied genotypes of FSHβ subunits. For the 1-year age group, significant increases in values (mean ± SE) of individual motility were detected in the AG genotype (70.5 ± 3.73) compared to values of AA (62.5 ± 4.82) and GG (60.19 ± 4.5) genotypes, which showed insignificant differences (P>0.05) in their values. The percentage of dead (17.19 ± 1.08) and abnormal sperms (6.72 ± 0.73) was lowest significantly (P<0.05) in the AG genotype in comparison with the values of AA (28.46 ± 1.5 and 14.39 ± 2.06, respectively) and GG genotypes (25.81 ± 2.03 and 13.55 ± 1.75, respectively) of FSHβ subunits respectively. However, the results of AA and GG genotypes showed no significant differences (P<0.05) in values of dead and abnormal sperms.

Table 2: Association of sperm properties and genotypes of FSHβ subunits in ram of 1-year age group rams.

Genotype	Sperm properties (%)
Genotype	Individual motility	Dead sperms	Abnormal sperms
AA	62.5 ± 4.82 B	28.46±1.5A	14.39 ± 2.06 A
GG	60.19 ± 4.5 B	25.81±2.03A	13.55 ± 1.75 A
AG	70.5 ± 3.73 A	17.19±1.08B	6.72 ± 0.73 B

Variation in large vertical letters refers to significant differences (P<0.05). AA= Adenine Adenine, GG= Guanine Guanine, AG= Adenine Guanine.

Sperm properties (%) and genotypes of FSHβ subunits in rams of more than one-year age group

For more than one year (>1year) age group, Table 3 showed a significant increases (P<0.05) in values (mean ± SE) of individual motility were reported in AG (79.08 ± 3.52) genotype of FSHβ subunits in comparison with values of AA (67.31 ± 4.18) and GG (65.22 ± 3.94) genotypes both were showed no significant differences (P>0.05) in their values. Regarding the percentage of dead and abnormal sperms, respectively, values were a significantly decline in AG genotype (12.4 ± 1.26 and 5.15 ± 1.01) and increased significantly (P<0.05) in the AA (21.73 ± 2.03 and 12.98 ± 1.64, respectively) and GG (20.15 ± 1.51 and 12.45 ± 1.82, respectively) genotypes, which both showed insignificant variation (P>0.05) between their values.

Table 3: Association of sperm properties (%) and genotypes of FSHβ subunits in rams of more than one year (>1 year) age group.

Genotype	Sperm properties (%)
Genotype	Individual motility	Dead sperms	Abnormal sperms
AA	67.31 ± 4.18 B	21.73±2.03A	12.98 ± 1.64 A
GG	65.22 ± 3.94 B	20.15±1.51A	12.45 ± 1.82 A
AG	79.08 ± 3.52 A	12.4±1.26B	5.15 ± 1.01 B

Variation in large vertical letters refers to significant differences (P<0.05). AA= Adenine Adenine, GG= Guanine Guanine, AG= Adenine Guanine.

Seminal characteristics and genotypes of FSHβ subunits in rams of 2-years age group rams

The results in Table 4 revealed that the values of individual motility showed a significant elevation in the AG genotype group (87.29 ± 3.36) and a decrease in AA (77.45 ± 4.23) and GG (76.82 ± 4.19) genotypes. Concerning the percentage of dead and abnormal sperms, respectively, though the values of both AA and GG were recorded no significant differences between their values; the AG genotype group (6.98 ± 0.89 and 3.22 ± 0.54, respectively) showed a significant reduction in its values as compared with other groups.

Table 4: Association of seminal characteristics and genotypes of FSHβ subunits in rams of 2-years age group rams.

Genotype	Sperm properties (%)
Genotype	Individual motility	Dead sperms	Abnormal sperms
AA	77.45 ± 4.23 B	15.12 ± 1.64 A	8.99 ± 1.16 A
GG	76.82 ± 4.19 B	12.71 ± 1.33 A	9.34 ± 1.25 A
AG	87.29 ± 3.36 A	6.98 ± 0.89 B	3.22 ± 0.54 B

Variation in large vertical letters refer to significant differences (P<0.05). AA= Adenine Adenine, GG= Guanine Guanine, AG= Adenine Guanine.

DISCUSSION

The association analysis revealed that rams with the heterozygous AG genotype had significantly higher sperm individual motility (P<0.05) than GG and AA genotypes in ram of 2-years age-old (Table 4) as compared with other age groups (Tables 2 and 3), these result was in agreement with Yang et al. (2011), Iyer and Rajkumar (2019) and Pascal et al. (2023), consistent with previous reports that polymorphisms of the FSHβ subunit gene were significantly associated with individual motility. Ram fertility is significantly impacted by polymorphisms in genes linked to semen quality. These genes also influence important traits such as sperm count, motility, and morphology in other species (Abril-Parreño et al., 2023). The Follicle-Stimulating Hormone Beta Subunit (FSH-β) confers the distinct biological action of FSH and its specific interaction with the FSH receptor. In the testes’ seminiferous tubules, it promotes spermatogenesis, or the generation of sperm. It acts predominantly on Sertoli cells, which in turn support the meiosis of spermatocytes. Understanding male fertility requires an understanding of polymorphisms (variations) in the FSHβ gene, which can impact the hormone’s activity (Dalvi et al., 2018; Wu et al., 2023). Consistent with findings of Primasari et al. (2021) in bulls, where low genetic diversity in the FSHβ gene was associated with poor semen quality and fertility, our results suggest that polymorphisms in this gene may play a crucial role in regulating testicular function and sperm motility with different ages (1-year and >1-year old). The observed differences among genotypes in our study could reflect varying levels of FSHβ gene expression or receptor interaction efficiency. Therefore, maintaining adequate genetic variability at this locus may be essential for sustaining optimal reproductive performance in rams (Kim et al., 1988).

Numerous studies have shown that genetic factors influence reproduction and semen quality traits in bulls (Fonseca et al., 2020), goats (Alberto et al., 2018), and boars (Krupa et al., 2023). Ram sperm properties are a quantitative trait controlled by multiple genes. The identification of these genes using molecular techniques will ultimately lead to the development of novel tools for analyzing complex genetic traits, thereby improving the selection efficiency of rams (Salleh et al., 2018; Iyer and Rajkumar, 2019). There were many reports regarding the use of the candidate gene as a marker for semen quality and fertility in bulls (Ghorbani et al., 2017; Özbek et al., 2021). Dai et al. (2009) reported that FSH-β gene polymorphisms also affect semen quality and fertility. Our results indicate that by 2 years of age, rams undergo a cascade of morphological, physiological, and behavioral events leading to increased gonadal activity, so the result in this study showed AG genotype had a significantly higher than GG and AA genotype in sperm individual motility, and decreased in dead and abnormality (P<0.05) of ram 2-years age-old (Taylor et al., 2018; Camara et al., 2020). The β-subunit determines the biological specificity of gonadotropins, and the transcriptional differences of genes encoding the β-subunit directly affect the synthesis and secretion of hormones (Simoni et al., 2020; Wang et al., 2021).

The current results corroborate earlier studies that shown the crucial function of the FSHβ gene in controlling the fertility and quality of semen in all mammals. According to Kim et al. (2011), POBA cattle with limited genetic diversity of the FSHβ gene had lower fertility and semen quality, indicating that this locus is being influenced by selection pressures that affect reproductive performance. Also, according to research by Lamminen et al. (2005) and Grigorova et al. (2010), allele A of the FSHβ gene is associated with improved sperm motility and viability in both fresh and post-thawed semen. One possible explanation for this positive impact is that the encoded protein undergoes changes to its biological activity and hormonal function as a result of non-synonymous single nucleotide polymorphisms (SNPs). Consistent with these results, the present investigation showed that compared to rams with the GG and AA genotypes, AG genotypes had substantially smaller percentages of dead and defective spermatozoa and considerably better sperm individual motility. This indicates that variations in the FSHβ gene’s genetic code can influence spermatogenesis and testicular function by altering the interactions between FSH and receptors as well as the amounts of gene expression. Thus, it may be crucial to optimize reproductive efficiency and direct selection programs for better breeding rams by maintaining sufficient genetic variety at this locus.

Selection of breeding rams would be the prerequisite for getting better fertility, which can be accomplished through selecting some important reproductive traits (Li et al., 2023; Sa et al., 2024). There is a range of moderate to high heritability in sperm motility (Omolaoye et al., 2022). Accordingly, it is anticipated that enhanced fertility through assisted selective breeding would result from a deeper comprehension of the genomic architecture of complex semen-related variables. To improve breeds, it’s critical to find polymorphisms that can act as genetic markers for enhanced reproductive performance (Ramirez-Diaz et al., 2023).

Specifically, at 2 years of age, rams with the AG genotype of the FSHβ gene showed far better sperm motility and lower percentages of dead and deformed spermatozoa compared to rams with the GG and AA genotypes. Alterations to the Hypothalamic-Pituitary-Gonadal (HPG) Axis with Age: The secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) increases in sexually mature rams at 2 years of age, which improves spermatogenesis and amplifies the phenotypic manifestation of advantageous genotypes. Sperm motility and morphology are impacted by aging-related changes in gene expression that pertain to mitochondrial function, oxidative stress, lipid metabolism, and membrane dynamics. The quality of the semen may be impacted by these alterations’ interactions with the FSHβ genotype. Semen characteristics including motility and viability can be affected by changes in protein function caused by non-synonymous mutations in the FSHβ gene. Under the hormonal milieu that exists at 2 years of age, the AG genotype may give a functional benefit. This research confirms what previous research has shown, which is that sheep and other mammals exhibit semen quality attributes linked to genetic variants in the FSHβ gene. One example is the work of Liang et al. (2024), who demonstrated how gene polymorphisms affect mammalian fertility and sperm quality. The intricate genetic structure of male fertility was further demonstrated by the findings of Khan et al. (2024), who found strong associations between semen quality features and polymorphisms in genes such as FSHR and INHA. The frequency of the pattern is largely unknown, but Sustainable breeding efforts, especially in indigenous ram populations, require genetic variety maintenance, even though the AG genotype is a potential target for enhancing fertility.

CONCLUSION AND RECOMMENDATIONS

In conclusion, the AG genotype of the FSHβ gene is associated with superior semen quality in 2-year-old rams. This is likely due to an interaction between genetic factors and age-related physiological changes. These findings can guide efforts to improve sheep reproductive efficiency through selective breeding. Although the AG genotype represents a promising target for improving fertility, maintaining genetic diversity is essential for sustainable breeding programs, particularly in indigenous ram populations.

ACKNOWLEDGEMENT

The researchers would like to thank the College of Medicine, University of Fallujah for supporting this study by providing the animal house and scientific laboratories.

NOVELTY STATEMENT

The true novelty of this research lies not only in the “discovery” of a new gene but also in its practical application to improve a local breed by linking genetic variation to an important production trait, taking into account the age factor, thus providing an effective and accurate tool for breeding programs.

Author’s Contribution

The first author designed the study, supervised the experimental work, and contributed to data interpretation, Second author performed the laboratory analyses, collected samples, and carried out the molecular and statistical procedures, third author assisted in data analysis, prepared the figures and tables, and contributed to writing the initial draft of the manuscript and the Forth author as corresponding and contributed to data interpretation, critical revision of the manuscript, and improvement of the final scientific content. All authors read and approved the final version of the manuscript.

Ethical approval

This study was conducted in the Animal House of the College of Medicine, University of Fallujah, and was approved by the Ethical Committee (ethical approval number: 8) before commencing the research work. All information is anonymized and used for research only.

Generative AI and AI-assisted technology statement

Generative AI tools (such as ChatGPT) were used only to assist in minor language editing and improving the clarity of a few non-scientific sections of the manuscript (e.g., novelty statement). No AI tools were used to generate, analyze, or interpret scientific data, or to write the Introduction, Methods, Results, or Discussion. All content has been thoroughly reviewed and approved by the authors, who take full responsibility for the accuracy and integrity of the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Abril-Parreño L, Carthy TR, Keogh K, Štiavnická M, O’Meara C, Lonergan P, Kenny DA, Fair S (2023). Genome-wide association study reveals candidate markers related to field fertility and semen quality traits in Holstein-Friesian bulls. Animals, 17: 100841. https://doi.org/10.1016/j.animal.2023.100841

Alberto FJ, Boyer F, Orozco-terWengel P, Streeter I, Servin B, de Villemereuil P, Benjelloun, B., Librado, P., Biscarini, F., Colli, L., Barbato, M., Zamani, W., Alberti, A., Engelen, S., Stella, A., Joost, S., Ajmone-Marsan, P., Negrini, R., Orlando, L., Rezaei, H. R., Pompanon, F. (2018). Convergent genomic signatures of domestication in sheep and goats. Nat Commun., 9(1): 813. https://doi.org/10.1038/s41467-018-03206-y

Albrkaty HY (2024). Sheep ages recognition based on teeth images. Int. J. Innov. Sci. Res. Technol., 371: 214-241. https://doi.org/10.38124/ijisrt/IJISRT24SEP371

Andreeva M, Stefanov R (2020). Influence of the cryopreservation on the vitality of the sperm of the different breeds of rams. Tradit Mod Vet Med., 5(2): 26-30.

Banihani SA (2020). Effect of statin on semen quality characteristics. Andrologia. 52(6): e13592. https://doi.org/10.1111/and.13592

Barquero V, Roldan ERS, Soler C, Yániz JL, Camacho M, Valverde A (2021). Predictive capacity of boar sperm morphometry and morphometric sub-populations on reproductive success after artificial insemination. Animals (Basel), 11(4): 920. https://doi.org/10.3390/ani11040920

Camara PM, Steele H, Reese S, Kölle S (2020). Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting. Sci. Rep., 10(1): 16522. https://doi.org/10.1038/s41598-020-73592-1

Dai L, Zhao Z, Zhao R, Xiao, S., Jiang, H., Yue, X., Li, X., Gao, Y., Liu, J., Zhang, J. (2009). Effects of novel single nucleotide polymorphisms of the FSH beta-subunit gene on semen quality and fertility in bulls. Anim. Reprod. Sci., 114(1-3): 14-22. https://doi.org/10.1016/j.anireprosci.2008.08.021

Dalvi V, Ingawale M, Deshmukh S, Kuralkar S, Bankar P, Ali S, Ingole R. S., and Hajare S. W. (2018). Association of FSH beta Gene Polymorphism on Testicular Parameters and Semen Quality in HF and Jersey Crossbred Bulls. Int. J. Livest. Res., 8(6): 41-47. https://doi.org/10.5455/ijlr.20171113103829

Duan H, Xiao L, Ge W, Yang S, Jiang Y, Lv J, Hu, J., Zhang, Y., Zhao, X., Hua, Y. (2021). Follicle-stimulating hormone and luteinizing hormone regulate the synthesis mechanism of dihydrotestosterone in sheep granulosa cells. Reprod. Domest. Anim., 56(2): 292-300. https://doi.org/10.1111/rda.13837

Fonseca PAS, Suárez-Vega A, Cánovas A (2020). Weighted gene correlation network meta-analysis reveals functional candidate genes associated with high- and sub-fertile reproductive performance in beef cattle. Genes (Basel), 11(5): 543. https://doi.org/10.3390/genes11050543

George D, Mallery P (2019). IBM SPSS statistics 26 step by step: A simple guide and reference. 16th ed. London: Routledge. https://doi.org/10.4324/9780429056765

Ghorbani A, Moeini MM, Souri M, Hajarian H (2017). Influences of dietary selenium, zinc and their combination on semen characteristics and testosterone concentration in mature rams during breeding season. J. Appl. Anim. Res., 46(1): 813-819. https://doi.org/10.1080/09712119.2017.1406858

Goshme S, Banerjee S, Rekik M, Haile A, Yitagesu E, Getachew T (2020). Evaluation and characterization of semen quality in rams of Menz, Dorper and Awassi crosses in different seasons in Ethiopia. Livest. Res. Rural. Dev., 32: 180.

Grigorova M, Punab M, Poolamets O, Kelgo P, Ausmees K, Korrovits P, Vihljajev V, Laan M (2010). Increased prevalence of the -211 T allele of follicle stimulating hormone (FSH) β subunit promoter polymorphism and lower serum FSH in infertile men. J. Clin. Endocrinol. Metab., 95: 100–108. https://doi.org/10.1210/jc.2009-1010

Hu K, Sun W, Li Y, Zhang B, Zhang M, Guo C, Guo, C., Chang, H., Wang, X. (2020). Study on the Mechanism of Sarsasapogenin in Treating Precocious Puberty by Regulating the HPG Axis. Evid. Based Complement. Altern. Med., 2020: 1978043. https://doi.org/10.1155/2020/1978043

Iyer B, Rajkumar S (2019). Retraction notice to Genome sequence and comparative genomics of Rhizobium sp. Td3, a novel plant growth promoting phosphate solubilizing Cajanus cajan symbiont. Microbiol. Res., 221: 70. https://doi.org/10.1016/j.micres.2019.02.002

Johannisson A, Morrell JM, Ntallaris T (2024). A combination of biomarkers for predicting stallion sperm fertility. Vet. Res. Commun., 48(4): 2157-2169. https://doi.org/10.1007/s11259-024-10372-6

Joshi MJ, Upadhyay J, Deshpande PP, Sharma ML, Navathe CP (2010). Design of microcontroller based system for automation of streak camera. Rev. Sci. Instrum., 81(8): 085106. https://doi.org/10.1063/1.3462984

Jabulani Nkululeko Ngcobo, Khathutshelo Agree Nephawe, Ayanda Maqhashu and Tshimangadzo Lucky Nedambale (2020). Seasonal Variations in Semen Parameters of Zulu Rams Preserved at 10°C for 72 H During Breeding and NonBreeding Season. American Journal of Animal and Veterinary Sciences 2020, 15 (3): 226.239. https://doi.org/10.3844/ajavsp.2020.226.239

Khan MZ, Chen W, Naz S, Liu X, Liang H, Chen Y, Kou X, Liu Y, Ashraf I, Han Y, Peng Y, Wang C, Zahoor M (2024). Determinant genetic markers of semen quality in livestock. Front. Endocrinol., 15: 1456305. https://doi.org/10.3389/fendo.2024.1456305

Kim KE, Gordon DF, Maurer RA (1988). Nucleotide sequence of the bovine gene for follicle-stimulating hormone beta-subunit. DNA. 7(4): 227-233. https://doi.org/10.1089/dna.1988.7.227

Krupa E, Krupová Z, Žáková E, Bauer J, Moravčíková N, Vrtková I (2023). An assessment of the genetic parameters of boars’ reproductive traits. Genes (Basel), 14(11): 2003. https://doi.org/10.3390/genes14112003

Lamminen T, Jokinen P, Jiang M, Pakarinen P, Simonsen H, Huhtaniemi I (2005). Human FSHβ subunit gene is highly conserved. Mol. Hum. Reprod., 11(8): 601–605. https://doi.org/10.1093/molehr/gah198

Li Q, Chao T, Wang Y, Xuan R, Guo Y, He P, Zhang, L., Wang, J. (2024). Transcriptome analysis revealed the characteristics and functions of long non-coding RNAs in the hypothalamus during sexual maturation in goats. Front. Vet. Sci., 11: 1404681. https://doi.org/10.3389/fvets.2024.1404681

Li W, Mi S, Zhang J, Liu X, Chen S, Liu S, Feng X, Tang Y, Li Y, Liu L, Fang, L., Zhang, S., Yu, Y. (2023). Integrating sperm cell transcriptome and seminal plasma metabolome to analyze the molecular regulatory mechanism of sperm motility in Holstein stud bulls. J. Anim. Sci., 101: skad214. https://doi.org/10.1093/jas/skad214

Liang H, Chen W, Liu X, Han Y, Khan A, Wang C, Khan MZ (2024). Genetic polymorphisms in genes associated with mammalian semen quality traits: A review. Agriculture, 14: 2137. https://doi.org/10.3390/agriculture14122137

Madeja ZE, Podralska M, Nadel A, Pszczola M, Pawlak P, Rozwadowska N (2021). Mitochondria content and activity are crucial parameters for bull sperm quality evaluation. Antioxidants (Basel), 10(8): 1204. https://doi.org/10.3390/antiox10081204

Maquivar MG, Smith SM, Busboom JR (2021). Reproductive management of rams and ram lambs during the pre-breeding season in US sheep farms. Animals (Basel), 11(9): 2503. https://doi.org/10.3390/ani11092503

Miller Y, Robertson S, Sisson S (1975). the anatomy of the domestic animal. Available from: https://vetbooks.ir/sisson-and-grossmans-the-anatomy-of-the-domestic-animals-volume-i-5th-edition/.

Neyeloff JL, Fuchs SC, Moreira LB (2012). Meta-analyses and Forest plots using a microsoft excel spreadsheet: Step-by-step guide focusing on descriptive data analysis. BMC Res. Notes, 5: 52. https://doi.org/10.1186/1756-0500-5-52

Ngcobo JN, Nephawe KA, Maqhashu A, Nedambale TL (2020). Seasonal variations in semen parameters of Zulu rams preserved at 10°C for 72 h during breeding and non-breeding season. Am. J. Anim. Vet. Sci., 15(3): 226-239. https://doi.org/10.3844/ajavsp.2020.226.239

Obaid AH, Nawfal AJ, Mahmood AN (2022). Investigation the effects of melatonin on induction of fertile estrus in anestrus Female dogs. In: 2022 5th International Conference on Engineering Technology and its Applications (IICETA); Al-Najaf, Iraq. pp. 482-486. https://doi.org/10.1109/IICETA54559.2022.9888741

Omolaoye TS, Hachim MY, du Plessis SS (2022). Using publicly available transcriptomic data to identify mechanistic and diagnostic biomarkers in azoospermia and overall male infertility. Sci. Rep., 12: 2584. https://doi.org/10.1038/s41598-022-06476-1

Özbek M, Hitit M, Kaya A, Jousan FD, Memili E (2021). Sperm functional genome associated with bull fertility. Front. Vet. Sci., 8: 610888. https://doi.org/10.3389/fvets.2021.610888

Pascal C, Nechifor I, Florea MA, Pânzaru C, Simeanu D, Mierliță D (2023). Diet influence on sperm quality, fertility, and reproductive behavior in karakul of Botoșani rams. Agriculture, 13(11): 2168. https://doi.org/10.3390/agriculture13112168

Primasari A, Efendy J, Prihandini PW (2021). Polymorphism of follicle stimulating hormone beta sub-unit (FSH-β) gene as a molecular marker for reproductive status in Peranakan Ongole x Bali crossbred (POBA) cattle. IOP Conf. Ser. Earth Environ. Sci., 788(1): 012108. https://doi.org/10.1088/1755-1315/902/1/012052

Ramirez-Diaz J, Cenadelli S, Bornaghi V, Bongioni G, Montedoro SM, Achilli A, Capelli C, Rincon JC, Milanesi M, Passamonti MM, Colli, L., Barbato, M., Williams, J. L., Marsan, P. A. (2023). Identification of genomic regions associated with total and progressive sperm motility in Italian Holstein bulls. J. Dairy Sci., 106: 407–420. https://doi.org/10.3168/jds.2021-21700

Recchia K, Jorge AS, Pessôa LVF, Botigelli RC, Zugaib VC, de Souza AF, Martins, D. D. S., Ambrósio, C. E., Bressan, F. F., Pieri, N. C. G (2021). Actions and roles of FSH in germinative cells. Int. J. Mol. Sci., 22(18): 10110. https://doi.org/10.3390/ijms221810110

Rougier C, Hieronimus S, Panaïa-Ferrari P, Lahlou N, Paris F, Fenichel P (2019). Isolated follicle-stimulating hormone (FSH) deficiency in two infertile men without FSH β gene mutation: Case report and literature review. Ann. Endocrinol. (Paris), 80(4): 234-239. https://doi.org/10.1016/j.ando.2019.06.002

Sa P, Gòdia M, Lewis N, Lian Y, Clop A (2024). Genomic, transcriptomic and epigenomic analysis towards the understanding of porcine semen quality traits. Past, current and future trends. Anim. Reprod. Sci., 269: 107543. https://doi.org/10.1016/j.anireprosci.2024.107543

Saeed FF, Zaid NW (2019). Serum and testicular testosterone levels of ram lamb during puberty. Adv. Anim. Vet. Sci., 7(2): 92-95. https://doi.org/10.17582/journal.aavs/2019/7.2.92.95

Saleh WM, Abdul-Ameer A (2019). Evaluation of in vitro fertilization index by caudal spermatozoa capacitation with different heparin concentration in Iraqi sheep. Iraqi J. Vet. Med., 43(1): 171-182. https://doi.org/10.30539/iraqijvm.v43i1.488

Salleh SM, Mazzoni G, Løvendahl P, Kadarmideen HN (2018). Gene co-expression networks from RNA sequencing of dairy cattle identifies genes and pathways affecting feed efficiency. BMC Bioinf., 19(1): 513. https://doi.org/10.1186/s12859-018-2553-z

Savvulidi FG, Ptacek M, Malkova A, Beranek J, Stadnik L (2021). Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program. Czech J. Anim. Sci., 66(2): 55-64. https://doi.org/10.17221/226/2020-CJAS

Simoni M, Brigante G, Rochira V, Santi D, Casarini L (2020). Prospects for FSH treatment of male infertility. J. Clin. Endocrinol. Metab., 105(7): dgaa243. https://doi.org/10.1210/clinem/dgaa243

Tanga CM, Waweru JW, Tola YH, Onyoni AA, Khamis FM, Ekesi S, Paredes, J. C. (2021). Organic waste substrates induce important shifts in gut microbiota of black soldier fly (Hermetia illucens L.): Coexistence of conserved, variable, and potential pathogenic microbes. Front Microbiol., 12: 635881. https://doi.org/10.3389/fmicb.2021.635881

Taylor JF, Schnabel RD, Sutovsky P (2018). Identification of genomic variants causing sperm abnormalities and reduced male fertility. Anim. Reprod. Sci., 194: 57-62. https://doi.org/10.1016/j.anireprosci.2018.02.007

Touré A, Martinez G, Kherraf ZE, Cazin C, Beurois J, Arnoult C, Ray, P.F., Coutton, C. (2021). The genetic architecture of morphological abnormalities of the sperm tail. Hum. Genet., 140(1): 21-42. https://doi.org/10.1007/s00439-020-02113-x

Ulloa-Aguirre A, Zariñán T, Jardón-Valadez E, Gutiérrez-Sagal R, Dias JA (2018). Structure-function relationships of the follicle-stimulating hormone receptor. Front. Endocrinol. (Lausanne), 9: 707. https://doi.org/10.3389/fendo.2018.00707

Wang HQ, Zhang WD, Yuan B, Zhang JB (2021). Advances in the regulation of mammalian follicle-stimulating hormone secretion. Animals (Basel), 11(4): 1134. https://doi.org/10.3390/ani11041134

Wu S, Xie J, Zhong T, Shen L, Zhao Y, Chen L, Gan M, Zhang S, Zhu L, Niu L (2023). Genetic polymorphisms in ESR and FSHβ genes and their association with litter traits in large white pigs. Anim. Biotechnol., 34(9): 4713–4720. https://doi.org/10.1080/10495398.2023.2187405

Yang WC, Tang KQ, Yu JN, Zhang CY, Zhang XX, Yang LG (2011). Effects of MboII and BspMI polymorphisms in the gonadotropin releasing hormone receptor (GnRHR) gene on sperm quality in Holstein bulls. Mol. Biol. Rep., 38(5): 3411-3415. https://doi.org/10.1007/s11033-010-0450-2

Zaher DM, El-Gamal MI, Omar HA, Aljareh SN, Al-Shamma SA, Ali AJ, Zaib, S., Iqbal, J. (2020). Recent advances with alkaline phosphatase isoenzymes and their inhibitors. Arch. Pharm. (Weinheim), 353(5): e2000011. https://doi.org/10.1002/ardp.202000011