Colostrum induced passive immune transfer in lambs

Year 2023, , 80 - 88, 31.08.2023

Caner Övet

https://doi.org/10.30704/http-www-jivs-net.1335313

Cited By: 1

Abstract

During last decades, the production and consumption of small ruminant milk have been increased. As a result of this, sheep and goat farming have been developing and scientists are focused on clinical and feeding strategy researches on these animals. By the evolutionary challenges and adaptations, colostrum has a crucial role in immune complementation for litter. As a result of these challenges and adaptations neonatal life is more important especially in ruminants due to its lifetime effect and the future of livestock. The passive immune transfer is the main mechanism explained by biological evolution between the dam and lamb which is influenced by certain factors related to both dam and litter. Today the importance of passive immune transfer is well known for the future of livestock economy and animal welfare. In the literature, researchers are focused on correlation between colostrum quality (especially immunoglobulin concentration) and blood serum levels of immunoglobulin levels in newborns. The aims of the present review are to discuss data of recent studies and how passive immune transfer occurred in lambs as well as effecting factors and to supply new ideas to researchers.

Keywords

colostrum, passive immunity, sheep, lamb mortality

References

• Abdelqader, A., Irshaid, R., Tabbaa, M. J., Abuajamieh, M., Titi, H., & Al-Fataftah, A. R. (2017). Factors influencing Awassi lambs survivorship under fields conditions. Livestock Science, 199, 1-6.
• Abdel-Salam, Z. A., Abdel-Salam, S. A. M., Abdel-Mageed, I. I., & Harith, M. A. (2019). Evaluation of proteins in sheep colostrum via laser-induced breakdown spectroscopy and multivariate analysis. Journal of Advanced Research, 15, 19-25.
• Abegaz, S., Hegde, B. P., & Taye, M. (2011). Growth and physical body characteristics of Gumuz sheep under traditional management systems in Amhara Regional State, Ethiopia. Livestock Research for Rural Development, 23(5), 10.
• Agenbag, B., Swinbourne, A. M., Petrovski, K., & van Wettere, W. H. (2021). Lambs need colostrum: A review. Livestock Science, 251, 104624.
• Agenbag, B., Swinbourne, A. M., Petrovski, K., & van Wettere, W. H. (2023). Validation of a handheld refractometer to assess Merino ewe colostrum and transition milk quality. Journal of Dairy Science, 106(2), 1394-1402.
• Ahmed, A., Egwu, G. O., Garba, H. S., & Magaji, A. A. (2010). Studies on risk factors of mortality in lambs in Sokoto, Nigeria. Nigerian Veterinary Journal, 31(1), 56-65.
• Alves, A. C., Alves, N. G., Ascari, I. J., Junqueira, F. B., Coutinho, A. S., Lima, R. R., ... & Abreu, L. R. (2015). Colostrum composition of Santa Inês sheep and passive transfer of immunity to lambs. Journal of Dairy science, 98(6), 3706-3716.
• Banchero, G. E., Clariget, R. P., Bencini, R., Lindsay, D. R., Milton, J. T., & Martin, G. B. (2006). Endocrine and metabolic factors involved in the effect of nutrition on the production of colostrum in female sheep. Reproduction nutrition development, 46(4), 447-460.
• Banchero, G. E., Milton, J. T. B., Lindsay, D. R., Martin, G. B., & Quintans, G. (2015). Colostrum production in ewes: a review of regulation mechanisms and of energy supply. Animal, 9(5), 831-837.
• Barza, H.; Marinescu, M.; Blaga, L. (1993) Disorders of foals 0–10 days of age. Part I. Revista Romana de Medicina Veterinara, 3, 9-20.
• Berge, A. C., Hassid, G., Leibovich, H., Solomon, D., & Haines, D. M. (2018). A field trial evaluating the health and performance of lambs fed a bovine colostrum replacement. Journal of animal research and nutrition, 3, 1-6
• Berhan, A., & Van Arendonk, J. (2006). Reproductive performance and mortality rate in Menz and Horro sheep following controlled breeding in Ethiopia. Small Ruminant Research, 63(3), 297-303.
• Brujeni, G. N., Jani, S. S., Alidadi, N., Tabatabaei, S., Sharifi, H., & Mohri, M. (2010). Passive immune transfer in fat-tailed sheep: Evaluation with different methods. Small Ruminant Research, 90(1-3), 146-149.
• Burezq, H. A., & Khalil, F. (2022). Improved vaccination protocol to enhance immunity in lambs of Kuwait farms. Iraqi Journal of Veterinary Sciences, 36(2), 539-548.
• Castellaro, G., Ochoa, I., Borie, C., & Parraguez, V. H. (2022). Effects of Strategic Supplementation with Lupinus angustifolius and Avena sativa Grains on Colostrum Quality and Passive Immunological Transfer to Newborn Lambs. Animals, 12(22), 3159.
• Castro, I., Alba, C., Aparicio, M., Arroyo, R., Jiménez, L., Fernández, L., ... & Rodríguez, J. M. (2019). Metataxonomic and immunological analysis of milk from ewes with or without a history of mastitis. Journal of dairy science, 102(10), 9298-9311.
• Castro, N., Capote, J., Alvarez, S., & Argüello, A. (2005). Effects of lyophilized colostrum and different colostrum feeding regimens on passive transfer of immunoglobulin G in Majorera goat kids. Journal of Dairy Science, 88(10), 3650-3654.
• Castro-Alonso, A., Castro, N., Capote, J., Morales-delaNuez, A., Moreno-Indias, I., Sánchez-Macias, D., ... & Argüello, A. (2008). Apoptosis regulates passive immune transfer in newborn kids. Journal of Dairy Science, 91(5), 2086-2088.
• Chniter, M., Salhi, I., Harrabi, H., Khorchani, T., Lainé, A. L., Nowak, R., & Hammadi, M. (2016). Physiological changes in the peri-partum period and colostral IgG transfer in prolific D’man sheep: effects of parity and litter size. Tropical Animal Health and Production, 48, 387-394.
• Constable, P. D., Hinchcliff, K. W., Done, S. H., & Grünberg, W. (2016). Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs and goats. Elsevier Health Sciences.
• Constantin, N. T., & Sipos, A. (2021). Passive transfer of immunoglobulıns from ewe to lamb. Scientific Works. Series C, Veterinary Medicine, 67(1).
• Cuttance, E. L., Regnerus, C., & Laven, R. A. (2019). A review of diagnostic tests for diagnosing failure of transfer of passive immunity in dairy calves in New Zealand. New Zealand Veterinary Journal, 67(6), 277-286.
• de Sousa, I. V. P., Silva, C. B., & Ribeiro, C. V. (2018). Influence of the birth order on the total solids concentration of frozen colostrum from Santa Inês ewes. In 55a Reunião Anual da Sociedade Brasileira de Zootecnia, 28° Congresso Brasileiro de Zootecnia, Goiânia, Brasil, 27 a 30 de agosto de 2018. Sociedade Brasileira de Zootecnia-SBZ, Associação Brasileira dos Zootecnistas.
• Demis, C., Aydefruhim, D., Wondifra, Y., Ayele, F., Alemnew, E., & Asfaw, T. (2020). Maternal Immunoglobulin in the Serum of Newborn Lambs and Its Relation With Neonatal Mortality. ,Journal of Animal and Feed Research, 10(3), 119-124.
• Dewell, R. D., Hungerford, L. L., Keen, J. E., Laegreid, W. W., Griffin, D. D., Rupp, G. P., & Grotelueschen, D. M. (2006). Association of neonatal serum immunoglobulin G1 concentration with health and performance in beef calves. Journal of American Veterinary Medical Association, 228(6), 914-921.
• dos Santos, JDC., Saraiva, EP., Pimenta Filho, EC., Neta, GCX., Morais, LKC., Teti, HS., & Fidelis, SS. (2023). Risk factors for neonatal mortality in sheep farming systems in tropical semi-arid regions. Journal of Agricultural Science, 1-12.
• Drikic, M., Windeyer, C., Olsen, S., Fu, Y., Doepel, L., & De Buck, J. (2018). Determining the IgG concentrations in bovine colostrum and calf sera with a novel enzymatic assay. Journal of animal science and biotechnology, 9, 1-9.
• DuBourdieu, D. (2019). Colostrum antibodies, egg antibodies and monoclonal antibodies providing passive immunity for animals. Nutraceuticals in Veterinary Medicine, 245-257.
• Elitok, B. (2018). Indicators of passive immunity failure in neonatal calves. Oncol Res Rev, 1(3), 1-2.
• Elsohaby, I., McClure, J. T., Hou, S., Riley, C. B., Shaw, R. A., & Keefe, G. P. (2016). A novel method for the quantification of bovine colostral immunoglobulin G using infrared spectroscopy. International Dairy Journal, 52, 35-41.
• Erdoğan, H. M. (2009). An epidemiological study on neonatal lamb health. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 15(2). 225-236.
• Fischer, A. J., Villot, C., van Niekerk, J. K., Yohe, T. T., Renaud, D. L., & Steele, M. A. (2019). Invited Review: Nutritional regulation of gut function in dairy calves: From colostrum to weaning. Applied Animal Science, 35(5), 498-510.
• Gokce, E., & Atakisi, O. (2019). Interrelationships of serum and colostral IgG (passive immunity) with total protein concentrations and health status in lambs. Kafkas Universitesi Veteriner Fakultesi Dergisi, 25(4), 387-396. Gokce, E., AH, K., Atakisi, O., & HM, E. (2013b). Risk factors associated with passive immunity, health, birth weight and growth performance in lambs: III. The relationship among passive immunity, birth weight gender, birth type, parity, dam. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 19(5), 741-747.
• Gokce, E., Atakisi, O., Kirmizigul, A. H., Erdogan, H. M. (2013a). Risk factors associated with passive immunity, health, birth weight and growth Performance in Lambs: II. Effects of Passive Immunity and Some Risk Factors on Growth Performance During the First 12 Weeks of Life. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 19(4). 619-627.
• Haenlein, G. F. W. (2001). Past, present, and future perspectives of small ruminant dairy research. Journal of dairy science, 84(9), 2097-2115.
• Harmon, R. S., & Senesi, G. S. (2021). Laser-induced breakdown spectroscopy–a geochemical tool for the 21st century. Applied Geochemistry, 128, 104929.
• Hernández-Castellano, L. E., Almeida, A. M., Ventosa, M., Coelho, A. V., Castro, N., & Argüello, A. (2014b). The effect of colostrum intake on blood plasma proteome profile in newborn lambs: low abundance proteins. BMC Veterinary Research, 10, 1-9.
• Hernández-Castellano, L. E., Suárez-Trujillo, A., Martell-Jaizme, D., Cugno, G., Argüello, A., & Castro, N. (2015). The effect of colostrum period management on BW and immune system in lambs: from birth to weaning. Animal, 9(10), 1672-1679.
• Hernandez-Castellano, L., M Almeida, A., Castro, N., & Arguello, A. (2014a). The colostrum proteome, ruminant nutrition and immunity: a review. Current Protein and Peptide Science, 15(1), 64-74.
• Herndon, C. N., Shanthalingam, S., Knowles, D. P., Call, D. R., & Srikumaran, S. (2011). Comparison of passively transferred antibodies in bighorn and domestic lambs reveals one factor in differential susceptibility of these species to Mannheimia haemolytica-induced pneumonia. Clinical and Vaccine Immunology, 18(7), 1133-1138.Higaki, S., Nagano, M., Katagiri, S., & Takahashi, Y. (2013). Effects of parity and litter size on the energy
• contents and immunoglobulin G concentrations of Awassi ewe colostrum. Turkish Journal of Veterinary & Animal Sciences, 37(1), 109-112.
• Holmøy, I. H., Kielland, C., Stubsjøen, S. M., Hektoen, L., & Waage, S. (2012). Housing conditions and management practices associated with neonatal lamb mortality in sheep flocks in Norway. Preventive Veterinary Medicine, 107(3-4), 231-241.
• Hunter, A. G., Reneau, J. K., & Williams, J. B. (1977). Factors affecting IgG concentration in day-old lambs. Journal of Animal Science, 45(5), 1146-1151.
• Ibrahim, N. H., Badawy, M. T., Zakzouk, I. A., & Younis, F. E. (2020). Kids’ survivability as affected by their body weight, blood biochemical indices and maternal and kids’ behavior in baladi and shami goats under semi-arid condition. World’s Veterinary Journal, 10(1), 105-117.
• Kessler, E. C., Bruckmaier, R. M., & Gross, J. J. (2019). Immunoglobulin G content and colostrum composition of different goat and sheep breeds in Switzerland and Germany. Journal of dairy science, 102(6), 5542-5549.
• Kessler, E. C., Bruckmaier, R. M., & Gross, J. J. (2021). Comparative estimation of colostrum quality by Brix refractometry in bovine, caprine, and ovine colostrum. Journal of dairy science, 104(2), 2438-2444.
• Lee, S. H., Jaekal, J., Bae, C. S., Chung, B. H., Yun, S. C., Gwak, M. J., ... & Lee, D. H. (2008). Enzyme‐linked immunosorbent assay, single radial immunodiffusion, and indirect methods for the detection of failure of transfer of passive immunity in dairy calves. Journal of veterinary internal medicine, 22(1), 212-218.
• Lopreiato, V., Ceniti, C., Trimboli, F., Fratto, E., Marotta, M., Britti, D., & Morittu, V. M. (2017). Evaluation of the capillary electrophoresis method for measurement of immunoglobulin concentration in ewe colostrum. Journal of Dairy Science, 100(8), 6465-6469.
• Loste, A., Ramos, J. J., Fernández, A., Ferrer, L. M., Lacasta, D., Verde, M. T., ... & Ortín, A. (2008). Effect of colostrum treated by heat on immunological parameters in newborn lambs. Livestock Science, 117(2-3), 176-183.
• Mandal, A., Prasad, H., Kumar, A., Roy, R., & Sharma, N. (2007). Factors associated with lamb mortalities in Muzaffarnagari sheep. Small Ruminant Research, 71(1-3), 273-279.
• Massimini, G., Britti, D., Peli, A., Cinotti, S. (2006) Effect of Passive Transfer Status on Preweaning Growth Performance in Dairy Lambs. Journal of the American Veterinary Medical Association, 229, 111-115.
• McGovern, F. M., Campion, F. P., Lott, S., & Boland, T. M. (2015). Altering ewe nutrition in late gestation: I. The impact on pre-and postpartum ewe performance. Journal of animal science, 93(10), 4860-4872.
• Moreno-Indias, I., Sánchez-Macías, D., Castro, N., Morales-delaNuez, A., Hernández-Castellano, L. E., Capote, J., & Argüello, A. (2012). Chemical composition and immune status of dairy goat colostrum fractions during the first 10 h after partum. Small Ruminant Research, 103(2-3), 220-224.
• Morittu, V. M., Lopreiato, V., Ceniti, C., Spina, A. A., Minuti, A., Trevisi, E., ... & Trimboli, F. (2020). Capillary electrophoresis as a rapid test for the quantification of immunoglobulin G in serum of newborn lambs. Journal of dairy science, 103(7), 6583-6587.
• Morris, S. T. (2017). Overview of sheep production systems. In Advances in sheep welfare (pp. 19-35). Woodhead Publishing.
• Morton, S. U., & Brodsky, D. (2016). Fetal physiology and the transition to extrauterine life. Clinics in perinatology, 43(3), 395-407.
• Mukasa-Mugerwa, E., Lahlou-Kassi, A., Anindo, D., Rege, J. E. O., Tembely, S., Tibbo, M., & Baker, R. L. (2000). Between and within breed variation in lamb survival and the risk factors associated with major causes of mortality in indigenous Horro and Menz sheep in Ethiopia. Small Ruminant Research, 37(1-2), 1-12.
• Muñoz, C., Carson, A. F., McCoy, M. A., Dawson, L. E. R., O’Connell, N. E., & Gordon, A. W. (2008). Nutritional status of adult ewes durig early and mid-pregnancy. 1. Effects of plane of nutrition on ewe reproduction and offspring performance to weaning. Animal, 2(1), 52-63.
• Nordi, W. M., Moretti, D. B., Lima, A. L., Pauletti, P., Susin, I., & Machado-Neto, R. (2012). Intestinal IgG uptake by small intestine of goat kid fed goat or lyophilized bovine colostrum. Livestock Science, 144(3), 205-210.
• Nouri, M., Zarrin, M., Ahmadpour, A., Castro, N., González-Cabrera, M., & Hernández-Castellano, L. E. (2023). Feed restriction around parturition does not affect colostrum immunoglobulin G concentration in dairy fat-tailed sheep but does affect performance and blood metabolites in newborn lambs. Journal of Dairy Science, 106(4), 2980-2988.
• Nowak, R., & Poindron, P. (2006). From birth to colostrum: early steps leading to lamb survival. Reproduction Nutrition Development, 46(4), 431-446.
• Nowak, R., Porter, R. H., Lévy, F., Orgeur, P., & Schaal, B. (2000). Role of mother-young interactions in the survival of offspring in domestic mammals. Reviews of Reproduction, 5(3), 153-163.
• Övet, C. (2023). Cytokines and growth factors in goat colostrum: a short review. Journal of Bahri Dagdas Animal Research, 12(1), 89-97.
• Pekcan, M., Fidanci, U. R., Yuceer, B., & Ozbeyaz, C. (2013). Estimation of passive immunity in newborn calves with routine clinical chemistry measurements. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 60(2), 85-88.
• Piwczyński, D., Sitkowska, B., & Wiśniewska, E. (2012). Application of classification trees and logistic regression to determine factors responsible for lamb mortality. Small Ruminant Research, 103(2-3), 225-231.
• Rodríguez, C., Castro, N., Capote, J., Morales-delaNuez, A., Moreno-Indias, I., Sánchez-Macías, D., & Argüello, A. (2009). Effect of colostrum immunoglobulin concentration on immunity in Majorera goat kids. Journal of Dairy Science, 92(4), 1696-1701.
• Sallam, A. M. (2019). Risk factors and genetic analysis of pre-weaning mortality in Barki lambs. Livestock Science, 230, 103818.
• Shiels, D., Loughrey, J., Dwyer, C. M., Hanrahan, K., Mee, J. F., & Keady, T. W. (2021). A survey of farm management practices relating to the risk factors, prevalence, and causes of lamb mortality in Ireland. Animals, 12(1), 30.
• Silva, S. R., Sacarrão-Birrento, L., Almeida, M., Ribeiro, D. M., Guedes, C., González Montaña, J. R., Pereira, A. F., Zaralis, K., Geraldo, A., Tzamaloukas, O., Cabrera, M. G., Castro, N., Argüello, A., Hernández-Castellano, L. E., Alonso-Diez, Á. J., Martín, M. J., Cal-Pereyra, L. G., Stilwell, G., & de Almeida, A. M.  (2022). Extensive sheep and goat production: The role of novel technologies towards sustainability and animal welfare. Animals, 12(7), 885.
• Sjoberg, A., & Van Saun, R. J. (2021). Use of brix refractometer in assessing sheep colostrum. In American Association of Bovine Practitioners Conference Proceedings (pp. 273-273).
• Sol Morales, M., Palmquist, D.L., Weiss, W.P. (2000): Milk fat composition on Holstein and Jersey cows with control or depleted copper status and fed whole soybeans or tallow. Journal of Dairy Science 83(9), 2112-2119.
• Strusińska, D., Mierzejewska, J., Skok, A. (2004): Concentration of mineral components β-carotene, vitamins A and E in cow colostrum and milk when using mineralvitamin supplements. Medycyna Weterynaryjna 60(2), 202-206. Swarnkar, C. P., Gowane, G. R., Prince, L. L. L., & Sonawane, G. G. (2019). Risk factor analysis for neonatal lamb mortality in Malpura sheep. Journal of Animal Sciences 89(6), 640-644.
• Tabatabaei, S., Nikbakht, G., Vatankhah, M., Sharifi, H., & Alidadi, N. (2013). Variation in colostral immunoglobulin G concentration in fat tailed sheep and evaluation of methods for estimation of colostral immunoglobulin content. Acta Veterinaria Brno, 82(3), 271-275.
• Talore, G. D. (2009). On-farm performance evaluation of indigenous sheep and goats in Alaba, Southern Ethiopia, Doctoral dissertation, Hawassa University.
• Taye, M., Abebe, G., Gizaw, S., Lemma, S., Mekoya, A., & Tibbo, M. (2010). Growth performances of Washera sheep under smallholder management systems in Yilmanadensa and Quarit districts, Ethiopia. Tropical Animal Health and Production, 42, 659-667.
• Tibbo, M., Mukasa-Mugerwa, E., Woldemeskel, M., & Rege, J. E. O. (2003). Risk factors for mortality associated with respiratory disease among Menz and Horro sheep in Ethiopia. The veterinary journal, 165(3), 276-287.
• Tizard, I. R. (2017). Veterinary Immunology-E-Book. Elsevier Health Sciences. Todaro, M., Maniaci, G., Gannuscio, R., Pampinella, D., & Scatassa, M. L. (2023). Chemometric approaches to analyse the composition of a ewe’s colostrum. Animals, 13(6), 983.
• Torres-Rovira, L., Pesantez-Pacheco, J. L., Hernandez, F., Elvira-Partida, L., Perez-Solana, M. L., Gonzalez-Martin, J. V., ... & Astiz, S. (2017). Identification of factors affecting colostrum quality of dairy Lacaune ewes assessed with the Brix refractometer. Journal of Dairy Research, 84(4), 440-443.
• Turkson, P. K., & Sualisu, M. (2005). Risk factors for lamb mortality in Sahelian sheep on a breeding station in Ghana. Tropical Animal Health and Production, 37, 49-64.
• Turner, J. R. (2009). Intestinal mucosal barrier function in health and disease. Nature Reviews Immunology, 9(11), 799-809.
• Vatankhah, M. (2013). Relationship Between Immunoglobulin Concentrations in The Ewe's Serum and Colostrum, and Lamb's Serum in Lori‐Bakhtiari Sheep. Iranian Journal of Applied Animal Science, 3(3), 539-544.
• Vatankhah, M., & Talebi, M. A. (2009). Genetic and non-genetic factors affecting mortality in Lori-Bakhtiari lambs. Asian-Australasian Journal of Animal Sciences, 22(4), 459-464.
• Viola, I., Tizzani, P., Perona, G., Lussiana, C., Mimosi, A., Ponzio, P., & Cornale, P. (2022). Hazelnut skin in ewes’ diet: Effects on colostrum immunoglobulin g and passive transfer of immunity to the lambs. Animals, 12(22), 3220.
• Wesemann, D. R., Portuguese, A. J., Meyers, R. M., Gallagher, M. P., Cluff-Jones, K., Magee, J. M., ... & Alt, F. W. (2013). Microbial colonization influences early B-lineage development in the gut lamina propria. Nature, 501(7465), 112-115.
• Wu, H., Xu, C., Wang, J., Hu, C., Ji, F., Xie, J., ... & Lv, R. (2023). Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant Activity and Hormone Levels, and Gene Expression in Mammary Tissue of Lactating Sows. Animals, 13(9), 1536.
• Yalçın, E., & Temizel, E. M. (2010). Sütten Kesme Öncesi Dönemde Oğlakların Büyüme Performansına Pasif Transfer Durumunun Etkisi. Uludağ Üniversitesi Veteriner Fakültesi Dergisi, 29(1), 23-26.
• Yang, Y., Zhao, X., Huang, D., Wang, J., Qi, Y., Jiang, L., Zhao, H. & Cheng, G. (2019). Changes in intestinal proteins induced by colostrum uptake in neonatal calves: Analysis by two-dimensional gel electrophoresis-based proteomics analysis. Animal Production Science, 59(8), 1483-1490.
• Yenilmez, K., Arslan, S., Kılıç, S., & Atalay, H. (2021). The effect of twinship on mineral matter, immunoglobulin G and lamb birth weight in late pregnant ewes and their newborn lambs. Van Veterinary Journal, 32(2), 62-68.
• Zhu, H. L., Zhao, X. W., Han, R. W., Du, Q. J., Qi, Y. X., Jiang, H. N., ... & Yang, Y. X. (2021). Changes in bacterial community and expression of genes involved in intestinal innate immunity in the jejunum of newborn lambs during the first 24 hours of life. Journal of Dairy Science, 104(8), 9263-9275.
• Zohary, D., Tchernov, E., & Horwitz, L. K. (1998). The role of unconscious selection in the domestication of sheep and goats. Journal of Zoology, 245(2), 129-135.