Thermo-physiological and Molecular Profiling of Two Indigenous Purebred Saudi Sheep under Acute Heat Stress Conditions

E. M. Samara, M. A. Bahadi, M. A. Khan, M. A. Al-Badwi, K. A. Abdoun, M. Afzal, S. S. Alghamdi, A. A. Al-Haidary

Abstract

In light of the escalating global concern regarding adaptation and resilience to elevated temperatures due to climate change, this experiment was designed to assess the thermo-physiological attributes of two native sheep breeds (Najdi and Naimi) and to delineate potential genetic factors conferring heat tolerance amidst acute exposure to elevated ambient temperatures. Meteorological and thermo-physiological parameters were scrutinized at distinct intervals (0 min, 30 min, 120 min, 24 hr, and 48 hr), alongside the analysis of heat-responsive gene expression at 0 min, 30 min, and 120 min, following the exposure of nine healthy male lambs from each breed (mean body weight: 25 kg; age: 4 months) to a bio-meteorologically-simulated environment, maintaining an average ambient temperature of 45 °C (approximately 93 units in the temperature-humidity index). In addition, blood samples were collected from each lamb, with total RNA isolated and purity assessed, followed by qRT-PCR analysis of 16 heat stress candidate genes using validated primers and standardized thermocycling protocols, including controls to ensure accuracy. Data were analyzed using statistical methods, including PROC GLM and PROC MEANS in SAS, one-way ANOVA, and pairwise differences with the LSD test for significance, while gene expression differences were calculated using the comparative Ct method and 2^ (−ΔΔCt) for relative quantification. The findings elucidate that the Najdi breed manifests heightened thermotolerance relative to the Naimi breed, as evidenced by diminished indicators of heat stress, encompassing skin temperature, respiratory rate, packed cell volume, adaptability coefficient, serum total protein, glucose levels, and triiodothyronine concentration. Moreover, analysis of gene expression patterns revealed widespread activation of heat stress-responsive genes in both breeds under thermal stress conditions; however, Najdi lambs consistently exhibited elevated expression levels of these genes compared to their Naimi counterparts. Notably, genes including HSP90AB1, HSPB6, HSF1, STIP1, HSP60, HSP90, and HSPB1 demonstrated particularly pronounced upregulation in Najdi lambs. In conclusion, the integrative thermo-physiological and molecular profiling highlights the superior thermotolerance and evolutionary adaptation of the Najdi breed to the hot climate of the KSA, in contrast to the Naimi breed.

References

Abdoun, K. A., E. M. Samara, A. B. Okab, & A. A. Al-Haidary. 2012. A comparative study on seasonal variation in body temperature and blood composition of camels and sheep. J. Anim. Vet. Adv. 11:769–763. https://doi.org/10.3923/javaa.2012.769.773
Agnew, L. L. & I. G. Colditz. 2008. Development of a method of measuring cellular stress in cattle and sheep. Vet. Immunol. Immunopathol. 123:197–204. https://doi.org/10.1016/j.vetimm.2008.01.038
Al-Haidary, A. A., R. S. Aljumaah, M. A. Alshaikh, E. M. Samara, K. A. Abdoun, A. B. Okab, & M. M. Alfuraiji. 2012. Thermoregulatory and physiological responses of Najdi sheep exposed to environmental heat load prevailing in Saudi Arabia. Pak. Vet. J. 32:515–519.
Al-Haidary, A. A., Y. Al-Dosari, A. Abd-Elwahab, E. M. Samara, M. A. Al-Badwi, & K. A. Abdoun. 2021. White hair coat color does not influence heat tolerance of sheep grazing under a hot arid environment. Small Rumin. Res. 201:106410. https://doi.org/10.1016/j.smallrumres.2021.106410
Ammar, M. H., A. M. Khan, H. M. Migdadi, S. M. Abdelkhalek, & S. S. Alghamdi. 2017. Faba bean drought responsive gene identification and validation. Saudi J. Biol. Sci. 24:80–89. https://doi.org/10.1016/j.sjbs.2016.05.011
Andersson, B. E. & H. Jónasson. 2006. Regulação da Temperatura e Fisiologia Ambiental. In: M. J. Swenson & W. O. Reece (eds.). Dukes –Fisiologia dos Animais Domésticos. 12th ed. Guanabara Koogan S. A., Rio de Janeiro. pp. 805–813.
Bai, H., H. Ukita, M. Kawahara, T. Mitani, E. Furukawa, Y. Yanagawa, N. Yabuuchi, H. Kim, & M. Takahashi. 2020. Effect of summer heat stress on gene expression in bovine uterine endometrial tissues. Anim. Sci. J. 91:e13474. https://doi.org/10.1111/asj.13474
Bharati, J., S. S. Dangi, S. R. Mishra, V. S. Chouhan, V. Verma, O. Shankar, M. K. Bharti, A. Paul, D. K. Mahato, G. Rajesh, G. Singh, V. P., Maurya, S. Bag, P. Kumar, & M. Sarkar. 2017. Expression analysis of Toll like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure. J. Therm. Biol. 65:48–56. https://doi.org/10.1016/j.jtherbio.2017.02.002
Brown-Brandl, T. M. 2018. Understanding heat stress in beef cattle. Rev. Bras. Zootecn 47:e20160414. https://doi.org/10.1590/rbz4720160414
Charoensook, R., K. Gatphayak, A. R. Sharifi, C. Chaisongkram, B. Brenig, & C. Knorr. 2012. Polymorphisms in the bovine HSP90AB1 gene are associated with heat tolerance in Thai indigenous cattle. Trop. Anim. Health Prod. 44:921–928. https://doi.org/10.1007/s11250-011-9989-8
Corazzin, M., E. Saccà, G. Lippe, A. Romanzin, V. Foletto, F. Da Borso, & E. Piasentier. 2020. Effect of heat stress on dairy cow performance and on expression of protein metabolism genes in mammary cells. Animals 10:2124. https://doi.org/10.3390/ani10112124
da Silva, R. G. & A. S. C. Maia. 2013. Principles of Animal Biometeorology. Springer, New York. https://doi.org/10.1007/978-94-007-5733-2
Dangi, S. S., M. Gupta, D. K. Maurya, V. P. Yadav, R. P. Panda, G. Singh, N. H. Mohan, S. K. Bhure, B. C. Das, S. Bag, R. Mahapatra, G. T. Sharma, & M. Sarkar. 2012. Expression profile of HSP genes during different seasons in goats (Capra hircus). Trop. Anim. Health. Prod. 44:1905–1912. https://doi.org/10.1007/s11250-012-0155-8
Dangi, S. S., S. K. Dangi, V. S. Chouhan, M. R. Verma, P. Kumar, G. Singh, & M. Sarkar. 2016. Modulatory effect of betaine on expression dynamics of HSPs during heat stress acclimation in goat (Capra hircus). Gene 575:543–550. https://doi.org/10.1016/j.gene.2015.09.031
Darling, N. J. & S. J. Cook. 2014. The role of MAPK signaling pathways in the response to endoplasmic reticulum stress. Biochim. Biophys Acta 1843:2150-2163. https://doi.org/10.1016/j.bbamcr.2014.01.009
El-Zarei, M. F., A. M. Alseaf, A. A. Alhaidary, E. F. Mousa, A. B. Okab, E. M. Samara, & K. A. Abdoun. 2019. Short-term heat shock proteins 70 and 90 mRNA expression profile and its relation to thermo-physiological parameters in goats exposed to heat stress. Int. J. Biometeorol. 63:459–465. https://doi.org/10.1007/s00484-019-01677-2
Fonsêca, V. F. C., A. S. C. Maia, E. P. Saraiva, C. C. M. Costa, R. G. da Silva, K. A. Abdoun, A. A. Al-Haidary, E. M. Samara, & A. Fuller. 2019. Bio-thermal responses and heat balance of a hair coat sheep breed raised under an equatorial semi-arid environment. J. Therm. Biol. 84:83–91. https://doi.org/10.1016/j.jtherbio.2019.05.024
Fuller, A., D. Mitchell, S. K. Maloney, & R. S. Hetem. 2016. Towards a mechanistic understanding of the responses of large terrestrial mammals to heat and aridity associated with climate change. Clim. Change Rep. 3:1–19. https://doi.org/10.1186/s40665-016-0024-1
Gaughan, J. B., V. Sejian, T. L. Mader, & F. R. Dunshea. 2018. Adaptation strategies: Ruminants. Anim. Front. 9:47-53. https://doi.org/10.1093/af/vfy029
Ghanem, A. M., L. S. Jaber, M. A. Said, E. K. Barbour, & S. K. Hamadeh. 2008. Physiological and chemical responses in water-deprived Awassi ewes treated with vitamin C. J. Arid Environ. 72:141–149. https://doi.org/10.1016/j.jaridenv.2007.06.005
Gorostizaga, A., L. Brion, P. Maloberti, F. C. Maciel, E. J. Podestá, & C. Paz. 2005. Heat shock triggers MAPK activation and MKP-1 induction in Leydig testicular cells. Biochem. Biophys. Res. Commun. 327:23–28. https://doi.org/10.1016/j.bbrc.2004.11.129
Grewal, S., A. Aggarwal, & M. N. Alhussien. 2021. Seasonal alterations in the expression of inflammatory cytokines and cortisol concentrations in periparturient sahiwal cows. Biol. Rhythm. Res. 52:1229–1239. https://doi.org/10.1080/09291016.2019.1670971
Haire, A., J. Bai, X. Zhao, Y. Song, G. Zhao, A. Dilixiati, J. Li, W. Sun, P. Wan, X. Fu, & A. Wusiman. 2022. Identifying the heat resistant genes by multi-tissue transcriptome sequencing analysis in Turpan Black sheep. Theriogenology 179:78–86. https://doi.org/10.1016/j.theriogenology.2021.11.008
Kalds, P., S. Zhou, Y. Gao, B. Cai, S. Huang, Y. Chen, & X. Wang. 2022. Genetics of the phenotypic evolution in sheep: A molecular look at diversity-driving genes. Genet. Sel. Evol. 54:61. https://doi.org/10.1186/s12711-022-00753-3
Kamboh, A. A., S. Q. Hang, M. Bakhetgul, & W. Y. Zhu. 2013. Effects of genistein and hesperidin on biomarkers of heat stress in broilers under persistent summer stress. Poult. Sci. 92:2411–2418. https://doi.org/10.3382/ps.2012-02960
Kelly, C. F. & T. E. Bond. 1971. Bioclimatic Factors and their Measurement: A Guide to Environmental Research on Animals. Natl. Acad. Sci., Washington, DC.
Kim, N. K., D. Lim, S. H. Lee, Y. M. Cho, E. W. Park, C. S. Lee, B. S. Shin, T. H. Kim, & D. Yoon. 2011. Heat shock protein b1 and its regulator genes are negatively correlated with intramuscular fat content in the longissimus thoracis muscle of hanwoo (Korean cattle) steers. J. Agric. Food Chem. 59:5657–64. https://doi.org/10.1021/jf200217j
Kregel, K. C. 2002. Heat shock proteins: Modifying factors in physiological stress responses and acquired thermotolerance. J. Appl. Physiol. 92:2177–2186. https://doi.org/10.1152/japplphysiol.01267.2001
Kumar, R., I. D. Gupta, A. Verma, N. Verma, & M. R. Vineeth. 2017. Single nucleotide polymorphisms in Heat Shock Protein (HSP) 90AA1 gene and their association with heat tolerance traits in Sahiwal cows. Indian J. Anim. Res. 51:64–69.
Kumar, R., I. D. Gupta, A. Verma, S. Singh, R. Kumari, & N. Verma. 2021. Genetic polymorphism in HSPB6 gene and their association with heat tolerance traits in Indian Karan Fries (Bos aurus x Bos indicus) cattle. Anim. Biotechnol. 29:1–12. https://doi.org/10.1080/10495398.2021.1899939
Li, Y., L. Kong, M. Deng, Z. Lian, Y. Han, B. Sun, Y. Guo, G. Liu, & D. Liu. 2019. Heat stress-responsive transcriptome analysis in the liver tissue of Hu sheep. Genes 100:395. https://doi.org/10.3390/genes10050395
Livak, K. J. & T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−deltadeltaCt method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lu, Z., M. Chu, Q. Li, M. Jin, X. Fei, L. Ma, L. Zhang, & C. Wei. 2019. Transcriptomic analysis provides novel insights into heat stress responses in sheep. Animals 9:387. https://doi.org/10.3390/ani9060387
Luna-Ramirez, R. I., S. W. Limesand, R. Goyal, A. L. Pendleton, G. Rincón, X. Zeng, G. Luna-Nevárez, J. R. Reyna-Granados, & P. Luna-Nevárez. 2023. Blood transcriptomic analyses reveal functional pathways associated with thermotolerance in pregnant ewes exposed to environmental heat stress. Genes 14:1590. https://doi.org/10.3390/genes14081590
Marai, I. F. M., A. A. El-Darawany, A. Fadiel, & M. A. M. Abdel-Hafez. 2007. Physiological traits as affected by heat stress in sheep – A review. Small Rumin. Res. 71:1–12. https://doi.org/10.1016/j.smallrumres.2006.10.003
Martins Júnior, L. M., A. P. R. Costa, D. M. R. Azevedo, S. H. N. Turco, J. E. G. Campelo, & M. C. S. Muratori. 2007. Adaptabilidade de caprinos Boer e Anglo-nubiana às condições climáticas do meio-norte do Brasil. Arch. Zootec. 56:103–113.
Matsumoto, T., M. Urushido, H. Ide, M. Ishihara, K. Hamada-Ode, Y. Shimamura, K. Ogata, K. Inoue, Y. Taniguchi, & T. Taguchi. 2015. Small heat shock protein beta-1 (HSPB1) is upregulated and regulates autophagy and apoptosis of renal tubular cells in acute kidney injury. PLoS One 10:e0126229. https://doi.org/10.1371/journal.pone.0126229
McManus, C., G. R. Paludo, H. Louvandini, R. Gugel, L. C. B. Sasaki, & S. R. Paiva. 2009. Heat tolerance in Brazilian sheep: Physiological and blood parameters. Trop. Anim. Health. Prod. 4:95–101. https://doi.org/10.1007/s11250-008-9162-1
McManus, C. M., C. M. Lucci, A. Q. Maranhão, D. Pimentel, F. Pimentel, & S. R. Paiva. 2022. Response to heat stress for small ruminants: Physiological and genetic aspects. Livest. Sci. 263:105028. https://doi.org/10.1016/j.livsci.2022.105028
Mishra, S. R. 2021. Behavioural, physiological, neuro-endocrine and molecular responses of cattle against heat stress: An updated review. Trop. Anim. Health Prod. 53:400. https://doi.org/10.1007/s11250-021-02790-4
Nguyen, A. N. & K. Shiozaki. 1999. Heat-shock-induced activation of stress MAP kinase is regulated by threonine- and tyrosine-specific phosphatases. Genes Dev. 13:1653-63. https://doi.org/10.1101/gad.13.13.1653
Parsell, D. A. & S. Lindquist. 1993. The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27:437–496. https://doi.org/10.1146/annurev.ge.27.120193.002253
Pugh, D. G., N. N. Baird, M. Edmondson, & T. Passler. 2020. Sheep, Goat, and Cervid Medicine-E-Book. Elsevier Health Sciences. https://doi.org/10.1016/C2017-0-02021-9
Raslan, F., T. Schwarz, S. Meuth, M. Austinat, M. Bader, T. Renné, K. Roosen, G. Stoll, A.L. Sirén, & C. Kleinschnitz. 2010. Inhibition of bradykinin receptor B1 protects mice from focal brain injury by reducing blood-brain barrier leakage and inflammation. J. Cereb. Blood Flow Metab. 30:1477–1486. https://doi.org/10.1038/jcbfm.2010.28
Rawash, R. A. A., M. A. Sharaby, & G. Hassan. 2022. Expression profiling of HSP 70 and interleukins 2, 6 and 12 genes of Barki sheep during summer and winter seasons in two different locations. Int. J. Biometeorol. 66:2047–2053. https://doi.org/10.1007/s00484-022-02339-6
Renaudeau, D., A. Collin, S. Yahav, V. de Basilio, J. L. Gourdine, & R. J. Collier. 2012. Adaptation to hot climate and strategies to alleviate heat stress in livestock production. Animals 6:707–728. https://doi.org/10.1017/S1751731111002448
Rhoad, A. O. 1944. The Iberia heat tolerance test for cattle. Trop. Agric. 21:162–164.
Rong, Y., M. Zeng, X. Guan, K. Qu, J. Liu, J. Zhang, H. Chen, B. Huang, & C. Lei. 2019. Association of HSF1 genetic variation with heat tolerance in Chinese cattle. Animals 9:1027. https://doi.org/10.3390/ani9121027
Rout, P., R. Kaushik, & N. Ramachandran. 2016. Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period. Cell Stress Chaperon. 21:645–651. https://doi.org/10.1007/s12192-016-0689-1
Sajjanar, B., R. Deb, U. Singh, S. Kumar, M. Brahmane, A. Nirmale, S. K. Bal, & P. S. Minhas. 2015. Identification of SNP in HSP90AB1 and its association with the relative thermotolerance and milk production traits in Indian dairy cattle. Anim. Biotechnol. 26:45–50. https://doi.org/10.1080/10495398.2014.882846
Samara, E. M., M. A. Bahadi, M. A. Al-Badwi, K. A. Abdoun, & A. A. Al-Haidary. 2023. A comparative thermophysiological study between two purebred Saudi sheep under biometeorologically-simulated environment. J. Saudi Soc. Agric. Sci. 22:283–287. https://doi.org/10.1016/j.jssas.2023.01.003
Samara, E. M., K. A. Abdoun, A. B. Okab, M. A. Al-Badwi, M. F. El-Zarei, A. M. Al-Seaf, & A. A. Al-Haidary. 2016. Assessment of heat tolerance and production performance of Aardi, Damascus, and their crossbred goats. Int. J. Biometeorol. 60:1377–1387. https://doi.org/10.1007/s00484-015-1131-6
Saudi National Center for Meteorology (SNCM). 2024. Historical Data Service. https://ncm.gov.sa/Ar/EService/met/Pages/ClimateDataRequest.aspx
Sejian, V., V. P. Maurya, & S. M. K. Naqvi. 2010. Adaptive capability as indicated by endocrine and biochemical responses of Malpura ewes subjected to combined stresses (thermal and nutritional) in a semi-arid tropical environment. Int. J. Biometeorol. 54:653–661. https://doi.org/10.1007/s00484-010-0341-1
Silanikove, N. 2000. Effects of heat stress on the welfare of extensively managed domestic ruminants: A review. Livest. Prod. Sci. 67:1–18. https://doi.org/10.1016/S0301-6226(00)00162-7
Singh, K. M., S. Singh, I. Ganguly, R. K. Nachiappan, A. Ganguly, R. Venkataramanan, A. Chopra, & H. K. Narula. 2017. Association of heat stress protein 90 and 70 gene polymorphism with adaptability traits in Indian sheep (Ovis aries). Cell Stress Chaperones 22:675–684. https://doi.org/10.1007/s12192-017-0770-4
Sivakumar, A. V. N., G. Singh, & V. P. Varshney. 2010. Antioxidants supplementation on acid base balance during heat stress in goats. Asian-Australas. J. Anim. Sci. 23:1462-1468. https://doi.org/10.5713/ajas.2010.90471
Sonna, L. A., J. Fujita, S. L. Gaffin, & C. M. Lilly. 2002. Effects of heat and cold stress on mammalian gene expression. J. Appl. Physiol. 92:1725–1742. https://doi.org/10.1152/japplphysiol.01143.2001
Starling, J. M. C., R. G. Silva, M. Cerón-Muñoz, G. S. S. C. Barbosa, & M. J. R. P. Costa. 2002. Analysis of some physiological variables for the evaluation of the degree of adaptation in sheep submitted to heat stress. Rev. Bras. Zootecn. 32:2070–2077. https://doi.org/10.1590/S1516-35982002000800022
Vihervaara, A. & L. Sistonen. 2014. HSF1 at a glance. J. Cell Sci. 127:261–266. https://doi.org/10.1242/jcs.132605
West, J. W. 2003. Effects of heat-stress on production in dairy cattle. J. Dairy Sci. 86:2131–2144. https://doi.org/10.3168/jds.S0022-0302(03)73803-X
Woolley, S. A., M. Salavati, & E. L. Clark. 2023. Recent advances in the genomic resources for sheep. Mamm. Genome. 34:545–558. https://doi.org/10.1007/s00335-023-10018-z
Yang, H., Y. L. Yang, G. Q. Li, Q. Yu, & J. Yang. 2021. Identifications of immune-responsive genes for adaptative traits by comparative transcriptome analysis of spleen tissue from Kazakh and Suffolk sheep. Sci. Rep. 11:3157. https://doi.org/10.1038/s41598-021-82878-x
Zeng, L., K. Qu, J. Zhang, B. Huang, & C. Lei. 2022. Genes related to heat tolerance in cattle-a review. Anim. Biotechnol. 15:1–9. https://doi.org/10.1080/10495398.2022.2047995

Authors

E. M. Samara
dremas@ksu.edu.sa (Primary Contact)
M. A. Bahadi
M. A. Khan
M. A. Al-Badwi
K. A. Abdoun
M. Afzal
S. S. Alghamdi
A. A. Al-Haidary
SamaraE. M., BahadiM. A., KhanM. A., Al-BadwiM. A., AbdounK. A., AfzalM., AlghamdiS. S., & Al-HaidaryA. A. (2024). Thermo-physiological and Molecular Profiling of Two Indigenous Purebred Saudi Sheep under Acute Heat Stress Conditions. Tropical Animal Science Journal, 47(3), 300-311. https://doi.org/10.5398/tasj.2024.47.3.300

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