UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL ULTRASONOGRAPHY B-MODE, ELASTOGRAPHY (ACOUSTIC RADIATION FORCE IMPULSE), COLOR DOPPLER AND HYSTEROSCOPY UTERINE IN POSTPARTUM IN SANTA INES SHEEP Renata Sitta Gomes Mariano Médica Veterinária 2018 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL ULTRASONOGRAPHY B-MODE, ELASTOGRAPHY (ACOUSTIC RADIATION FORCE IMPULSE), COLOR DOPPLER AND HYSTEROSCOPY UTERINE IN POSTPARTUM IN SANTA INES SHEEP Renata Sitta Gomes Mariano Orientador: Prof. Dr. Wilter Ricardo Russiano Vicente Coorientadores: Prof. Dr. Marcus Antonio Rossi Feliciano Prof. Dr. Pedro Paulo Maia Teixeira 2018 Tese apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para obtenção do título de Doutor em Medicina Veterinária área de Reprodução Animal. M333u Mariano, Renata Sitta Gomes Ultrasonography B-Mode, Elastography (Acoustic Radiation Force Impulse), Color Doppler and hysteroscopy uterine in postpartum in Santa Ines sheep / Renata Sitta Gomes Mariano. -- Jaboticabal, 2018 52 p. : il., tabs., fotos + 1 CD-ROM Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientadora: Prof. Dr. Wilter Ricardo Russiano Vicente 1. Puerpério. 2. Involução uterina. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. AUTHOR CURRICULUM DATA RENATA SITTA GOMES MARIANO – was born in Garça, São Paulo state, Brazil, on May 11th of 1990. In 2008 she joined the Faculty of Veterinary Medicine in Garça, concluding her academic experience on December 2012. In 2013, she began a Master student in Animal Reproduction Science, at Faculty of Agricultural and Veterinary Sciences – FCAV/Unesp in Jaboticabal under Prof. Dr. Wilter Ricardo Russiano Vicente. She concluded her Master’s degree and on 2015 she began her PhD at the same university, and field, under Dr. Vicente supervisor. Where in 2017 she moved to the College Station, Texas, United States, to join researches on fetal programming at Texas A&M University during four months in Animal Science department under Dr. Rodolfo C Cardoso. “I never lose. I either win or I learn.” (Nelson Mandela) “Though nobody can go back and make a new beginning, anyone can start over and make a new ending.” (Chico Xavier) To my Dad and Mom, with love. ACKNOWLEDGEMENTS This journey would not have been possible without the support, encouragement and help from many people. First, I give thanks to God for protection and ability to work, for giving me strength and wisdom to win and never give up on my dreams. To my family, for their love and support, and for being the best family I could ever ask for. Thank you for encouraging me in all of my pursuits, giving me strength to reach my goals, and inspiring me to chase my dreams. I am especially grateful to my parents, who supported me emotionally and financially. Thank you for the endless love, support and encouragement, without which I would never have enjoyed so many opportunities. I always knew that you believed in me and wanted the best for me. Thank you both for encouraging me to be a better person every day, and for always trusting and supporting my decisions. My gratitude to both of you is beyond words. I love you! To my brother who has been my best friend all my life, thank you for all your encouragement, advice and support over the years. A lovely thank to a person that has not been part of my life for too long but has changed everything. Thanks Carson Landers for being my best friend, supporting me during this period, being so lovely and patient, and for helping me through the good and bad, easy and hard moments. Thanks to Prof. Dr. Wilter, my advisor, for the continuous support during my PhD research, for his patience, motivation, knowledge, and especially for his confidence on me. I could not have imagined having a better guide and mentor throughout all my years in graduate school. It was an honor for me to share of his exceptional knowledge, but also his extraordinary human qualities. I express my thanks to my coadvisors, Prof. Dr. Marcus, and Prof. Dr. Pedro Paulo, for the academic support, thoughtful insight, advice, and guidance on the work presented in this thesis. To my research group and coworkers, I am truly thankful for prompt help, whenever I needed during my PhD project. Special thanks to Victor, Priscila, Mariana, Augusto, Marjury, Ana Paula, Michelle, Roberta, Daniele, Paulo Henrique, and Maria Eduarda. The general help and friendship were greatly appreciated. Thanks to all those sincere friends that I met in Jaboticabal during this journey. I owe a sense of gratitude to Ricardo Uscategui for the statistical analysis and interpretation of data, Felipe Barros for the corrections and suggestions to improve my thesis, and Luciana Nakaghi for all her help and guidance writing this thesis. To the animals, an essential part of this work, thanks for their valuable contribution to the science. I place my heartfelt thanks to Univ. Estadual Paulista "Júlio de Mesquita Filho", to the academic and technical support, to the staff and facilities of the Veterinary Hospital "Governador Laudo Natel" - FCAV / Unesp Jaboticabal. I am also grateful to Texas A&M University for the wonderful experience during my Sandwich Program, especially to Dr. Rodolfo Cardoso who accepted me as a visiting scholar, and to all my friends that I made in College Station. Thanks to National Council for Scientific and Technological Development for the financial support (CNPq – Grant 441492/2014-2). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. I gratefully acknowledge São Paulo Research Foundation (FAPESP) for the financial support (Process nº 2015/18519-8). Regrettably, but inevitably, the list will be incomplete, and I hope that those who have not been mentioned here, but directly or indirectly contributed to this project, forgive me and accept my sincere appreciation of their influence on my PhD. Renata i SUMMARY CHAPTER 1 – GENERAL CONSIDERATIONS ........................................................ 1 Introduction .............................................................................................................. 1 Literature review ....................................................................................................... 1 Anatomy and physiology of puerperium ................................................................... 2 Postpartum assessment by means of puerperium ................................................... 4 Conclusion ............................................................................................................... 7 References ............................................................................................................... 7 CHAPTER 2 – B-MODE, DOPPLER AND ACOUSTIC RADIATION FORCE IMPULSE (ARFI) ELASTOGRAPHY EVALUATION IN POSTPARTUM SHEEP .... 12 Abstract .................................................................................................................. 12 Introduction ............................................................................................................ 13 Material and Methods ............................................................................................. 14 Results ................................................................................................................... 17 Discussion .............................................................................................................. 22 Conclusion ............................................................................................................. 25 References ............................................................................................................. 26 CHAPTER 3 – POSTPARTUM EVALUATION IN EWE: STUDY OF UTERINE AND CERVICAL INVOLUTION BY HYSTEROSCOPY AND HISTOLOGY. .................. 30 Abstract .................................................................................................................. 30 Introduction ............................................................................................................ 31 Material and Methods ............................................................................................. 32 Results ................................................................................................................... 33 Discussion .............................................................................................................. 35 Conclusion ............................................................................................................. 37 References ............................................................................................................. 37 ii iii ULTRASONOGRAPHY B-MODE, ELASTOGRAPHY (ACOUSTIC RADIATION FORCE IMPULSE), COLOR DOPPLER AND HYSTEROSCOPY UTERINE IN POSTPARTUM IN SANTA INES SHEEP ABSTRACT – The aim of this study was to evaluate the uterine characteristics of Santa Inês sheep during the postpartum period, using mode B-mode ultrasonography, elastography ARFI (acoustic radiation force impulse), Doppler and hysteroscopy, with emphasis on the early diagnosis of reproductive alterations, evaluation of development and regression during this period. Twenty Santa Inês sheep were used and designated after clinical and obstetric evaluation. B-mode, Doppler and elastography ARFI evaluations were performed by transabdominal approach using the Siemens S2000 ultrasound system, with a multi-frequency and convex transducer of 5.0 to 8.0 MHz. Ultrasonography of the uterine structure was performed at immediate postpartum (M0) and sequentially every 48 hours, during 30 days, totaling 16 experimental samples. Ultrasonography characteristics of the uterus (echogenicity, echotexture, and biometry), vascular parameters (color Doppler) and stiffness aspects (qualitative and quantitative elastography) of the uterine structures were evaluated during the postpartum. Hysteroscopy evaluation of the uterine involution of the sheep was performed as follows: immediate moment after lambing (M0) and sequentially every 6 hours, until the moment where the endoscopic access to the uterus through the cervix was no longer possible. Uterine biopsy was performed at the same moments. The experimental design will be completely randomized, with a significance level of 5% for tests performed. Keywords: ovine, ultrasound, puerperium, uterus. iv LIST OF ABBREVIATIONS ARFI – Acoustic radiation force impulse SD – standard deviation % - percentage; Kg – kilogram; h – hour; M0 – immediate postpartum moment n – sample size pp – days postpartum SWV – Shear wave velocity v LIST OF TABLES Chapter 2 Table 1. Mean values (±SD) (m/s) for shear waves velocities (SWV) and depth of different moments (days pp) during the postpartum involution in healthy ewes using acoustic radiation force impulse (ARFI) quantitative elastography (Jaboticabal, 2018)...................................... 21 Chapter 3 Table 1. Correlation of number and percentage (%) of animals evaluated and time of cervical closure observed trough endoscopy during the postpartum involution in healthy ewes (Jaboticabal, 2018).................................................................................................... 34 vi LIST OF FIGURES Chapter 2 Figure 1. Graphic illustration showing the correlation between uterine measurements (uterine wall thickness; biometry of lumen uterine body) and postpartum days in healthy ewes uterus……………………………… 18 Figure 2. Ultrasound color Doppler image of uterine blood flow during postpartum in healthy ewes. A: 4 days; B: 30 days pp………………………………… 19 Figure 3. Ultrasound image of postpartum healthy ewe uterus during its qualitative ARFI elastography analysis. Note the B-mode image (left) and the image of the elastography (right) of the uterus showing a homogeneous and dark (hard) image, respectively.…........................... 20 Figure 4. Ultrasound Image of postpartum healthy ewe uterus during its quantitative ARFI elastography analysis. Note the measurement of the shear velocity of uterine wall, with the presence of a caliper for the portion assessed…................................................................................. 21 Chapter 3 Figure 1. Photomicrographs of the histological evaluation of ewes uterus in the postpartum period. A) Cervical region showing moderate hyperemia of the vessels in the submucosal and discrete neutrophilic inflammatory infiltrate. B) Uterine epithelium showing increased vascularization and marked neutrophilic inflammatory infiltrate. C) Endometrial region presenting the predominance of neutrophils in the inflammatory infiltrate. D) Endometrial glands with epithelial cells in apoptosis. Hematoxylin-Eosin.…………………………………………………………. 35 1 CHAPTER 1 – GENERAL CONSIDERATIONS 1. Introduction Puerperium is the period after completion of parturition and it occurs in a decreasing logarithmic scale, especially during the first week after lambing. After parturition, the uterus undergoes marked remodeling during involution; however, little is known about the hormonal, cellular and molecular mechanisms that regulate this process. Considering the requirement to establish early diagnostic methods for the evaluation of puerperal changes in sheep, monitoring of this period may be essential to avoid a decline in reproductive efficiency. There are conflicting reports regarding uterine involution in ewes, which may reflect differences in breeds and management. An understanding of the postpartum recovery process of the reproductive tract in the ewe is of increasing importance in production systems where more than pregnancy per year is desirable. It was hypothesized that the use of imaging techniques allows the monitoring of uterine involution and physiological changes during the postpartum period in ewes. In this study we review the puerperal period, the mechanisms underlying the ensuing of normal ovine reproduction, and the ultrasound assessment to evaluate this period in ewe as an experimental model or for application in veterinary medicine. 2. Literature Review Improving reproductive efficiency in domestic farm animals is critically important to the livestock industry, ewes must be breed within 30 to 40 days after parturition (Abecia et al., 2017). For intensive sheep production, the postpartum period is of most importance for the continuity of the reproductive function of females and may vary from 17 to 40 days (Zdunczyk et al., 2004; Fernandes et al., 2013), being influenced by breed, season, management, intercurrences during partum, number of births and age (Zdunczyk et al., 2004). Nutrition also is essential for reproductive efficiency of ruminants, affecting directly the duration of postpartum anestrus (Diskin et al., 2003; Rhind, 2004) The postpartum period has also a reproductive and economic importance that is influential in achieving a satisfactory interval between partums and impact the 2 number of offsprings produced per year (Sanchez et al., 2002), mainly under intensive accelerated production systems (Greyling, 2000; Ababneh and Degefa, 2005). Indeed, the influences that contribute to a low productivity in the herds are associated with delayed uterine involution due to inflammatory and/or infectious processes of the uterus, disorders in endometrial regeneration, anestrus and delay in the onset (Sheldon et al., 2017). In the prepartum, there are risks of physical damage during the birth process and also an upsurge of microbial infections in the ewe. Therefore, uterine involution may be delayed in case of occurrences of dystocia, number of fetus and abortions (Sheldon et al., 2006; Benzaquen et al., 2007), as well as uterine prolapse and placental retention among other disorders (Gomes et al., 2014). Regarding uterine pathologies, it is also important to mention endometritis, pyometra and hydrometra/mucometra (Medan and El-daek, 2015). Ultrasonography in small ruminants’ production has been increasing the routine applicability and play a role being responsible for expressive benefits to the reproductive management, enabling puerperal evaluation and consequent uterine involution of sheep in real time, and being a practical tool (Lohan et al., 2004; Hajurka et al., 2005). Actually, it is the only non-invasive techniques that may reveal details of the progressive changes in the uterus of ewes (Ioannidi, 2017). 2.1 Anatomy and Physiology of Postpartum The uterus of domestic animals consists in: the corpus or body and its endometrium, two horns and the uterine cervix. Uterine wall has three different layers: endometrium; myometrium, and perimetrium (Akers and Denbow, 2008). The perimetrium and the myometrium, share a similar echogenic structure, so an ultrasound differentiation is not possible. The uterine endometrium of ewes has both aglandular caruncular and glandular intercaruncular areas (Hafez, 2004; Wang et al., 2013). Myometrium is hormone sensitive and undergoes both hypertrophy and hyperplasia during pregnancy, progressively returning to its normal size during the postpartum period (Lowe and Anderson, 2015). Placentation in sheep (synepitheliochorial) involves growth and union of placental cotyledons with endometrial caruncles developing placentomes, which are the primary sites of conceptus-maternal exchange for gases and micronutrients, such as amino acids and 3 glucose (Bazer et al., 2012). The ovine cervix is a long, fibrous tubular composed predominantly of connective tissue with an outer serosal layer and inner luminal epithelium. The lumen is highly convoluted and tortuous due to the presence of cervical rings and provide a physical barrier to external contaminants (Kershaw et al., 2005). In ovines, gestation lasts on average 150 days and may vary due to interference of maternal, fetal and genetic factors. If this does not occur, a new estrous cycle will begin at an average interval of 17 days (Oliveira et al., 2013). During the beginning of pregnancy, the uterus is located in the pelvic cavity, similarly to non-pregnant status. In mid-gestation and later stages of gestation, it becomes an abdominal organ. After lambing and before subsequent gestation, four events must occur concomitantly: uterine involution, endometrial regeneration, elimination of bacterial contamination and return of ovarian cycling. The uterine involution begins immediately after lambing and involves physical shrinkage, necrosis and sloughing of caruncles, and also the regeneration of the endometrium. Sloughing of the uterine caruncles contributes to reduction in weight of the uterus because they take over half of the weight of the uterus. There is initially regeneration of the endometrium in the inter- caruncular areas and then by centripetal growth of the cells over the caruncle (Sheldon, 2004, 2008). As puerperium advances, the involution processes also involve regressive changes in the uterus, as endometrial regeneration, and cervix (Bajcsy, 2005). There is a progressive decreasing in vaginal discharge and in uterine and cervical diameters. It occurs due to vasoconstriction and peristaltic contractions (Leslie, 1983; Wehrend and Bostedt, 2003), remodeling of caruncles, regeneration of endometrial tissue, reduction in uterine blood flow and endometrial vascularity, as well as reduction of smooth muscle mass (Guilbault et al., 1984; Slama et al., 1991). The cervical canal is still widely opened immediately after birth, but elements of the soft birth canal still provide a certain degree of closing, because in a physiological situation the surrounding tissues and organs of the abdominal and pelvic cavity compress the cervix. A balanced and coordinated endocrine system is important for normal reproductive function, that is, the returning of ovarian cycling. This involves homeostasis among gonadotrophin releasing hormones (GnRH) from the 4 hypothalamus; follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL) from the adenohypophysis; prostaglandin F2-alpha (PGF2α) from the uterus and the gonadal steroids (Kota et al., 2013). After parturition, steroid hormone concentrations decrease to basal values and there is an increase in plasma FSH concentration within days of calving that stimulates the emergence of the first postpartum follicular wave (Sheldon, 2008). Thus, the ability to achieve maximum reproductive efficiency depends on a thorough understanding of postpartum hormonal changes. The postpartum fertility of sheep depends on uterine physiological involution and the resumption of cyclic ovarian activity (Lamraoui et al., 2017). However, reestablishment between the time of complete uterine involution and the return to cyclic activity in the postpartum period is not totally elucidated (Hayder and Ali, 2008; Nasciutti et al., 2011). 2.2 Postpartum assessment by means of ultrasonography To maximize sheep production, it is necessary to adopt tools that enable reproductive monitoring in order to improve reproductive performance and herd productivity (Sharkey et al., 2001). In small ruminants, due to the impossibility of performing rectal palpation, accurate evaluation of the internal organs of the reproductive tract becomes unreasonable (Oliveira et al., 2013). Uterine involution studies have revealed different intervals to complete this process. Most studies were assessed by hormonal dosages (Ishwar, 1995), radiography (Tian and Noakes, 1991), laparotomy (Rubianes et al., 1996) or at the moment of slaughter (Zdunczyk et al., 2004). The cons of the above techniques include invasiveness, reduced accuracy and difficulty to apply in clinical conditions (Ioannidi et al., 2017). In sheep, ultrasonography is routinely used for pregnancy diagnosis, but there is insufficient information utilizing this technique for the evaluation of the uterine regression in this species. It has been reported as a practical and efficient tool assessment the uterine involution during post-partum period in cows (Sheldon and Ownes, 2017), mares (Griffin and Ginther, 1991), goats (Ababneh and Degefa, 2005; Badawi et al., 2014; Fasulkov, 2014) and sheep (Hauser and Bostedt, 2002; Zdunczyk https://www.sciencedirect.com/science/article/pii/S0921448816303650#bib0145 https://www.sciencedirect.com/science/article/pii/S0921448816303650#bib0080 5 et al., 2004; Hayder and Ali, 2008; Nasciutti et al., 2011, Gomes et al., 2014; Ioannidi et al., 2017) Uterine characteristics that can be assessed by ultrasonography of the uterus during postpartum are asymmetry of the organ, distention of uterine lumen, presence, quantity and texture of uterine content, thickness of uterine wall, and localization of inflammatory on the uterine wall, texture of uterine wall, alterations in uterine wall vascularization and confirmation of uterine involution completion (Ioannidi et al., 2017). Ultrasonography has a role in differentiating the normal or abnormal uterus during postpartum (Medan and El-daek, 2015). It also provides additional information on physiological and pathological processes in the uterus, which may contribute to the development of new methods for the treatment of reproductive disorders in ovines (Jaśkowski et al., 2013). The ultrasound also offers possibilities to diagnose abnormalities in the uterine regression like accumulation of lochia in the uterine lumen or the presence of retained foetal membranes leading to a prolonged phase of involution, placental retention among other disorders and other such endometritis, hydrometra, uterine infection and hemorrhage (Hauser and Bostedt, 2002). Ultrasound examination of the female reproductive system in small ruminants can be conducted using two approaches: transabdominal and transrectal (Zdunczyk et al., 2004). To evaluate the uterine involution in sheep, it is recommended in the first week post-partum the transabdominal evaluation, because it is more sensitive than the transrectal, as the uterus remains in the cranioventral abdominal portion until the eighth postpartum day (Hauser and Bostedt, 2002). As the uterus involution is in progress, the use of transrectal ultrasonography permits more objective measurement of the uterine horns diameter and visualization of the uterine lumen (Sheldon, 2004). Uterus and its content in a normal uterus can be easily visualized during the early stage of the puerperium appearing enlarged, with a heterogeneous echotexture (Hauser and Bostedt, 2002), and diameter can be up to 10 cm (Fernandes et al., 2013). As the uterine size decreases progressively, it may be more difficult to image it. Ababneh and Degefa (2005) reported difficulty to image the uterus after the 13th day post-partum. Research reports evaluating by ultrasound the uterus during the postpartum period have shown different intervals to complete uterine involution, between 17-35 https://www.researchgate.net/scientific-contributions/2033493745_JM_Jaskowski 6 days postpartum (Ali et al., 2001; Hauser and Bostedt, 2002; Zdunczyk et al., 2004; Ababneh and Degefa, 2005; Badawi et al., 2014; Fasulkov, 2014; Medan and El-Daek, 2015; Elmetwally and Bollwein, 2017. Gomes et al. (2014) reported a reduction of uterine depth slower in ewes with twin parturition, as compared to singleton parturition, and a uterine depth decrease in all ewes during postpartum period, observing the sharpest drop from day 1 to day 16 after parturition, corresponding to more than 50% of total uterine regression. Accordingly, Hauser and Bostedt (2002) reported uterine size decreasing by 50% of its size, 5 days postpartum. However, Rubianes and Ungerfeld (1993) reported that 97% of the uterine involution in the animals evaluated occurred approximately day 17 postpartum. Other researchers found that this involution occurs in a longer period, at 28 days (Regassa and Noakes,1999) or between the fourth and fifth week postpartum (Hayder and Ali, 2008). According to Ababneh and Degefa (2005) the nonexistence of intrauterine fluid 4-7 days postpartum on ultrasound imaging demonstrate rapid regression of the uterus, and the involution of uterus may be characterized by a small cross-sectional diameter of the horns and absence of lochia in the uterus by ultrasonography (Zdunczyk et al., 2004). The ability to measure changes in blood flow during postpartum period provides a promising diagnostic tool for assessing the status of uterine involution during this period. It has been used for several years to identify postpartum uterine involution in women, reporting an association between uterine blood flow and delayed uterine involution (Mulic-Lutvica et al., 2007; Guedes-Martins et al., 2015). It has been used to assess uterine blood flow during the postpartum period in cows (Krueger, 2009; Heppelmann et al., 2013), sheep and goats (Elmetwally and Bollwein, 2017; Ioannidi et al., 2017). Preliminary finds in ewes have indicated that blood flow and diameter of the uterine artery decrease progressively, starting soon after lambing. Changes are of greater magnitude during the first post-partum week and the results became significant subsequently to the 20th day post-partum compared to findings immediately after lambing (Ioannidi et al. 2015). Elmetwally e Bollwein (2017) reported significantly decrease in uterine blood flow during the postpartum period in goats and sheep, especially during the first nine days postpartum, and these important changes can be explained because of loss of metabolic requirements of the fetus and placenta. 7 3. Conclusion The use of ultrasonography complement the monitoring of uterine involution in ewe, since uterine structures cannot be assessed by rectal or abdominal palpation in this species. This is a promissory tool to distinguish the pathological from the normal puerperium and thereby avoid unnecessary invasive procedures. Thus, the early diagnosis of intercurrences could improving the survival rate of obstetric patients and reproductive life, as well reducing the occurrence of infertile animals due to pathological postpartum. It is also important the knowledge obtained from ultrasonography examinations can help us better understanding the physiology of the postpartum period. 4. References Akers RM, Denbow DM (2008) Anatomy and Physiology of Domestic Animals. 2nd edition, Blackwell Publishing, Ames, Iowa; 504. Ababneh MM, Degefa T (2005) Ultrasonic assessment of puerperal uterine involution in balady goats. J Vet Med A Physiol Pathol Clin Med; 52:244-248. Abecia JA, Chemineau P, Gómez A, Palacios C, Keller M, Delgadillo JA (2017) Exposure to Photoperiod-Melatonin-Induced, Sexually-Activated Rams after Weaning Advances the Resumption of Sexual Activity in Post-Partum Mediterranean Ewes Lambing in January. Vet. Sci. 4:1-9. Ali A, Salem AA, El-Din Zain A (2001) Ultrasonographic assessment of postpartum uterine involution and onset of ovarian activity in the Ossimi ewe. Proceedings of the Egyptian Society for Animal Reproduction and Theriogenology. Badawi ME, Makawi SEA, Abdelghafar RM, Ibrahim MT (2014) Assessment of postpartum uterine involution and progesterone profile in nubian goats (Capra Hircus). J Adv Vet Anim Res; 1(2): 36-41. Bajcsy AC (2005) Physiological and clinical aspects of uterine contractility during the postpartum period in cows. Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University; 1–14. Bazer FW, Song G, Kim J, Dunlap KA, Satterfield MC, Johnson GA, Wu G (2012) Uterine biology in pigs and sheep. Journal of Animal Science and Biotechnology, 3(1), [23]. https://doi.org/10.1186/2050-7445-3-23 Diskin MG, Mackey DR, Roche JF, Sreenan JM (2003) Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Anim Reprod Sci; 78:345-370. 8 Elmetwally M, Bollwein H (2017) Uterine blood flow in sheep and goats during the peri- parturient period assessed by transrectal Doppler sonography. Anim Reprod Sci, 176:32-39. Fasulkov I (2014) Ultrasonography of uterine involution in goats. J Fac Vet Med; 40:63–69. Fernandes CES, Cigerza CF, Pinto GS, Miazi, C, Barbosa-Ferreira M, Martins, CF (2013) Características do parto e involução uterina em ovelhas nativas do pantanal brasileiro. Ciência Ani Bras; 14(2):245-252. Gomes MGT, Macedo-Júnior GL, Ferreira MIC, Borges I, Varago FC, Lago LA, Henry M (2014) Some aspects of the puerperium after singleton and twin parturitions in Santa Inês ewes submitted to energy restriction during pregnancy. Small Rumin Res; 120(2):219 – 223. Guedes-Martins L, Gaio AR, Saraiva J, Cunha A, Macedo F, Almeida H (2015) Uterine artery impedance during the first eight postpartum weeks; Scientific Reports. Guilbault LA, Thatcher WW, Foster DB, Caton D. Relationship of 15-keto-13, 14- dihydro-prostaglandin F2α concentrations in peripheral plasma with local uterine production of F series prostaglandins and changes in uterine blood flow during the early postpartum period of cattle (1984). Biol Reprod; 31:870–878. Greyling, JPC (2000) Reproduction traits in the boer goat doe. Small Rumin Res; 36(2):171–177. Hajurka J, Macak V, Hura V (2005) Influence of health status of reproductive organs on uterine involution in dairy cows. Bull Vet Inst Pulawy 49, 53-58. Hayder M, Ali A (2008) Factors affecting the postpartum uterine involution and luteal function of sheep in the subtropics. Small Rumin Res; 79:174-178. Hauser, B., Bostedt, H (2002) Ultrasonographic observations of uterine regression in the 286 ewe under different obstetrical conditions. J. Vet. Med; 49:511-516. Heppelmann M, Krueger L, Leidl S, Bollwein H (2013) Transrectal Doppler sonography of uterine blood flow during the first two weeks after parturition in cows. J Vet Sci;14:323–327. Ioannidi KS, Mavrogianni VS, Valasi I, Barbagianni MS, Vasileiou NGC, Amiridis GS, Fthenakis GC, Orfanou DC (2017) Ultrasonographic examination of the uterus of ewes during the post-partum period. Small Rumin. Res;152:74–85. Ishwar AK (1995) Pregnancy diagnosis in sheep and goats: a review. Small Rumin Res; 17:37-44. 9 Jaśkowski JM; Włodarek J; Gehrke M; et al. (2013) Use of Doppler ultrasonography in the reproduction of cows. Medycyna Weterynaryjna.69:585-591. Kershaw CM, Khalid M, McGowan, MR, Ingram K, Leethongdee S, Wax G, Scaramuzzi RJ (2005) The anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen. Theriogenology; 64:1225-1235. Kota SK, Gayatri K, Jammula S, et al. (2013) Endocrinology of parturition. Indian Journal of Endocrinology and Metabolism. 2013; 17:1:50-59. Kucharski J, Zezula-szpyra A, Doboszyńska T, Milewski S, Tański Z, Mercik L (1989) The study on the postpartum period in selected group of ewes in polish merino sheep. Clinical observations of the sexual organs. Pol Arch Wet; 29:201-210. Krueger L, Koerte J, Tsousis G, Herzog K, Flachowsky G, Bollwein H (2009) Transrectal Doppler sonography of uterine blood flow during the first 12 weeks after parturition in healthy dairy cows. Anim Reprod Sci;114:23–31. Lamraoui R, Farida A, Bouzebda Z (2017) Resumption of Ovarian Cyclicity During Postpartum in Winter-Lambing Ouled Djellal Ewes in Algerian Semi-Arid Area. Global Veterinaria; 18:27-30. Leslie KE (1983) The events of normal and abnormal postpartum reproductive endocrinology and uterine involution in dairy cows: a review. The Canad Vet J; 24(3):67-71. Lohan IS, Malik RK, Kaker ML (2004) Uterine involution and ovarian follicular growth during early postpartum period of murrah buffaloes (Bubalus bubalis). Asian-Aust J Anim Sci, 17(3):313-316. Lowe, J.S.; Anderson, P.G. Female Reproductive System. In: Stevens & Lowe's Human Histology (Fourth Edition). 2015; p.337-362 Medan MS, Al Daek T (2015) Uterine involution and progesterone level during the postpartum period in barbary ewes in north Libya. Open Vet J; 5(1):18-23. Mulic-Lutvica A, Axelsson O (2007) Postpartum ultrasound in women with postpartum endometritis, after cesarean section and after manual evacuation of the placenta. Acta Obst Gyn Scandi;86(2):210–217. Nasciutti NR, Oliveira RSBR, Silva NC, Franco MTF, Tsuruta SA, Ferreira IC, Sauti JPE (2011) Avaliação clínica da involução uterina em ovelhas da raça santa inês. Biosc J; 27:649-655. Okano A, Tomizuka T (1987) Ultrasonic observation of postpartum uterine involution in the cow. Theriogenology; 27(2):369-382. 10 Oliveira, MEF; Feliciano, MAR (2013) Ultrassonografia da Reprodução. In: Biotécnicas Reprodutivas em Ovinos e Caprinos. Primeira edição. São Paulo: Editora MedVet, p.121-146. Regassa F, Noakes DE (1999) Acute phase protein response of ewes and the release of PGFM in relation to uterine involution and the presence of intrauterine bacteria. Vet Rec, 144:502–506. Rhind SM (2004) Effects of maternal nutrition on fetal and neonatal reproductive development and function. Anim Reprod Sci, 82(83):169-181. Rubianes E, Ungerfeld R (1993) Uterine involution and ovarian changes during early postpartum in autumnlambing corriedale ewes. Theriogenology; 40:365- 372. Rubianes E, Ungerfeld R, Vifroles C, Carbajal B, De Castro T, Ibarra D (1996) Uterine involution time and ovarian activity in weaned and suciling J Anim Sci; 76:153-155. Sanchez MA, Garcia P, Menendez S, Sanchez B, Gonzalez M, Flores JM (2002) Fibroblastic growth factor receptor (FGF-R) expression during uterine involution in goat. Anim Reprod Sci; 69:25–35. Sharkey S, Callan RJ, Mortimer R, Kimberling C (2001) Reproductive techniques in sheep. Vet Clin North Am Food Anim Pract; 17, 435–455. Sheldon IM. The pospartum uterus (2004) Vet Clin Food Anim; 20:569-591. Sheldon IM, Lewis GS, Leblanc S, Gilbert RO (2006) Defining postpartum uterine disease in dairy cattle. Theriogenology; 65:1516-1530. Sheldon IM, Williams EJ, Miller ANA, Nash DM, Herath S (2008) Uterine diseases in cattle after parturition. The Veterinary Journal;176:1:115–121. Slama H, Vaillancourt D, Goff AK (1991) Pathophysiology of the puerperal period: Relationship between prostaglandin E2 (PGE2) and uterine involution in the cow. Theriogenology; 36:1071–1090. Tian W, Noakes DE (1991) A radiographic method for measuring the effect of exogenous hormone therapy on uterine involution in ewes. Vet Rec; 129:436-466. Wang Y, Wang C, Hou Z, Miao K, Zhao H, Wang R, Guo M, Wu Z, Tian J, An L (2013) Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period. J Anim Sci Biotechnol; 4:39. Wehrend A, Bostedt H (2003) Examinations on the incidence of cervical dystocia and disorders of cervical involution in the cow postpartum. Dsch Tieraerztl Wschr; 110:483-486. 11 Zdunczyk S., Milewski S, Barański W, Janowski T, Szczepański W, Jurczak A, Raś A, Lesnik M (2004) Postpartum uterine involution in primiparous and pluriparous polish longwool sheep monitored by ultrasonography. Bulletin Vet Inst Pulawy; 48:255-257. 12 CHAPTER 2 – B-MODE, DOPPLER AND ACOUSTIC RADIATION FORCE IMPULSE (ARFI) ELASTOGRAPHY EVALUATION IN POSTPARTUM SHEEP ABSTRACT – To evaluate the uterine involution by ultrasonography during postpartum of healthy ewe, twenty adult multiparous Santa Ines ewes were selected. Ultrasonography postpartum evaluation (B-mode, color Doppler and ARFI elastography) of the uterine structure was performed at immediate postpartum (M0) and sequentially every 48 hours, during 30 days, totaling 16 experimental samples. The echotexture did not present significant variations (p>0.05) remaining homogeneous in most evaluations during the postpartum period, and echogenicity of the uterus increased (p = 0.0452). A progressive and remarkable decrease of the uterine total diameter were observed (p<0.0001), especially during the first days postpartum. The measurement of uterine wall gradually decreased, as well the endometrial, myometrium and lumen diameter progressively decreased (p<0.0001) as the days progressed. Uterine blood flow was observed in all animals, and decreased during postpartum period, being significantly lower (p= 0.0225) on the 30th day postpartum. On qualitative elastography, the uterine parenchyma was not deformable and the images presented as homogeneous dark areas. On quantitative elastography, the mean shear velocity values of the uterine wall did not differ during the postpartum period. The B-mode, color Doppler and ARFI elastography of the uterus in healthy ewe could be easily performed. This is the first study that evaluate the stiffness of uterine wall parenchyma in animals, providing baseline data about quantitative e qualitative stiffness of the normal uterus, and may be a useful tool for the early diagnosis of uterine alterations during the postpartum period, using the reference parameter established for the assessment of uterine integrity during postpartum period. Thus, the early diagnosis of intercurrences could improving the survival rate of obstetric patients and reproductive life, as well reducing the occurrence of infertile animals due to pathological postpartum. Keywords: puerperium, uterine involution, uterine stiffness, sheep. 13 1. Introduction The postpartum period, also known as the puerperium, is defined as the time between parturition and completion of uterine involution (Sheldon and Owens, 2017; Sheldon, 2004). The uterine involution involves intense modifications as contraction of muscle fibers, catabolism, physical shrinkage, necrosis, sloughing of the caruncles and regeneration of the uterine ephithelium (Sheldon et al., 2008; Bajcsy, 2005). During involution, the size of the uterus diminishes and both the myometrium and endometrium are restored and so the position, then the uterus will be prepared for a next conception (Noakes, 2001). The physiological events that take place during the postpartum period influences next conception and pregnancy and provide a reproductive and economic importance that is significant in achieving a satisfactory interval between partums (Sanchez et al., 2002). Postpartum fertility in ewes depends on uterine physiological involution and restoration of cyclicity (Takayama et al., 2010). However, few information is available taking into account the reestablishment between the time of complete uterine involution and the return to cyclical activity (Hayder and Ali, 2008; Nasciutti et al., 2011). Thus, we believe monitoring postpartum period allows an early diagnosis of alterations, and an efficiently treatment of uterine diseases, to limit their negative effect on fertility. Ultrasonography is the greatest non-invasive technique, which may reveals details of the progressive changes in the uterus of ewes (Badawhi, 2014), plays a key role to distinguish the normal to abnormal postpartum uterus, and permits an objective measurement and visualization of the uterine horns and lumen diameter (Medan and El-Daec, 2015). The advantage of using non-invasive methods is that they can be applied under practical conditions, allowing analysis of either physiological or pathological events (Bajcsy, 2005). B-mode ultrasonography and color Doppler may be an useful tool to evaluate obstetrics and gynecology disorders, as well as uterine involution. Studies in humans describe the ability of Doppler to measure changes in uterine blood flow rates during the postpartum period, being a promising diagnostic tool for assessing uterine involution (Brackleyet al., 1998; Mulic-Lutvica et al., 2007; Guedes-Martinset al., 2015). 14 Elastography, a new technique based on ultrasonography recently been introduced in obstetrics and gynecology (Hernandez-Andrade et al., 2013), is used to evaluate stiffness in a tissue using a short acoustic push pulse in the target tissue (Karaman et al., 2016). This enable accurate assessment of stiffness intrauterine pathologies and it is consider a way to “imaging palpation” (Woźniak et al., 2016). The advantages of acoustic radiation force impulse (ARFI) elastography include the repeatability of objective measurements, and the ability to evaluate qualitative and quantitative information of tissue stiffness without requirement for external compression (Sporea et al., 2012; Alan et al., 2016). The elasticity of soft tissues is measured to investigate differential diagnosis of many diseases, such as inflammation, fibrosis, and tumoral tissues (Karaman et al, 2016; Tan et al., 2013). However, there are no elastographic studies of uterus during postpartum involution in ewes using the ARFI technique, which could provide even more physiologic and pathological information. Considering the necessity to carry out studies to establish the physiological patterns of postpartum that enable early diagnosis methods for evaluation of possible puerperal changes in sheep, monitoring this period becomes essential. This avoid a decline in reproductive efficiency, infertility in animals with high breeding values and delay in the return to cyclicity. The aim of this study was to describe the physiological changes throughout uterine involution, evaluating the uterine regression and tissue stiffness during postpartum involution by B mode ultrasonography, color Doppler and ARFI elastography to elucidate the mechanism of physiological uterine involution and uterine characterization during the postpartum period in ewe. 2. Material and methods Ethical aspects All animal procedures were approved by the Animal Ethics and Welfare Committee of the Faculty of Agricultural and Veterinary Sciences, Univ. Estadual Paulista (Unesp), Jaboticabal, Brazil (protocol N ° 12338/15). 15 Animals Twenty adult multiparous healthy Santa Ines ewes, aged 3.1 ± 1.1 years, weighing 45.4 ± 4.3 kg and exhibiting a mean body score of 3 (scale 1-5, Jefferies 1961), were selected for this study following clinical background evaluation, physical examinations, hematological tests and ultrasonography of the reproductive system. Ewes were maintained in an elevated sheep house at the Animal Reproduction Department, fed with corn silage, balanced commercial concentrate, mineral salt and water ad libitum during pregnancy and throughout the postpartum period. Only the animals that had non-intercurrent full-term normal delivery were used in this study. Experimental Protocol The animals were brought to the ultrasound laboratory, maintained in quadrupedal station, and no sedation was required throughout the entire duration of scanning. A wide trichotomy of the abdominal region was performed to facilitate the examinations and coupling gel applied thereupon. The ultrasonographic postpartum evaluations (B-mode, Doppler and elastography) of the uterine structure of the sheep was performed as follows: immediate moment (M0) and sequentially every 48 hours, during 30 days, totaling 16 experimental samples. B-mode and color Doppler ultrasonography Ultrasonography evaluations started on B-mode using the ACUSON S2000® (Siemens®, Munich, Germany) ultrasound system, and were performed by the same experienced operator using a convex multi-frequential transducer (4C1®; 1-4.5 MHz; Siemens®, Munich, Germany). The transducer was positioned on the right or left inguinal area. Imaging starts by locating the bladder, which were used as an acoustic window to facilitate examination, especially after the first week postpartum. Images were obtained on the longitudinal and the transverse ultrasonographic planes. When uterus was located, the characteristics of the uterine wall such as ecotexture (homogeneous or heterogeneous) and echogenicity (hypoechoic, hyperechoic or isoechoic compared to the adjacent tissues, or with a mixed appearance), thickness of the uterine layers (myometrium and endometrium), diameter of lumen and uterine 16 body, and characteristics of uterine contents (anechoic or hypoechogenic with or without cellular debris) were evaluated. Color Doppler were used to determine the characteristics in uterine blood flow such as presence or absence of vascularization, type of flow (arterial, venous or turbulent and mixed) and type of vessel (peripheral, central, or diffuse), as well as flow changes during the postpartum period ARFI elastography Following B-mode ultrasound, same portions studied were considered to accomplished analysis of the elastographic parameters, and a specific software designed for qualitative and quantitative image analyses (Virtual Touch Tissue Quantification® - VTTQ; Siemens®, Munich, Germany) was used. For the adjustment of the quantitative elastographic technique and the values for the shear wave velocity (SWV) of the uterine tissue, the caliper was placed within the uterine wall (endometrium/myometrium), and a minimum of three samples to obtain the mean, with a depth ranging from 0.5 to 5.0 cm were obtained in each portion evaluated. The values of the quantitative evaluation were expressed in the shear wave velocity (SWV - m/s). Each tissue stiffness evaluated qualitatively by elastogram grayscale image of this studied was calculated by analyzing the relative displacements of tissue elements due to an acoustic pressure pulse. For the interpretation of the formed elastographic image, white regions indicated less rigid tissue (more elastic/softer or more deformable regions) than the dark regions (more rigid structures/harder/not deformable). Statistical analysis This is a descriptive observational study with repeated measures. Statistical analysis was performed using the software R, version 3.3.0 (R® foundation for statistical computing, Austria). Data were tested initially for normality (Shapiro test) and homoscedasticity of variances (Barlett test). The variables resulting from the different analyzes were compared between the times by analysis of variance (ANOVA) and Tukey's post test, or by the Kruskal Wallis test and Dunns post-test otherwise. Correlation studies (Spearman or Pearson) and regression (linear and non-linear) were performed in relation to the moments. The qualitative characteristics were compared 17 by the Chi-square test. Differences were considered significant when p-value < 0.05 (5%). 3. Results All animals included in this study had a normal birth with no obstetrical complications. Uterine ultrasonography could be performed in all animals (n=20), and the uterine characteristics were determined without any difficulties in all animals evaluated by transabdominal approach. No intercurrences were observed during the experimental period and the animals showed no signs of discomfort during exams. Some qualitative variables varied during the involution process in relation to time. The uterine echogenicity increased, turn into hypoechoic then isoechoic (p = 0.0452), and after 20 days of partum hyperechoic echogenicity was no longer observed. The echotexture did not present significant variations (p>0.05) remaining homogeneous in most evaluations during the postpartum period. In the present study, the uterine contents were present in all animals until the 8th day postpartum (pp), and decreased (p=0.0215) gradually by the 22nd day pp, when it was no longer observed. The uterine contents were hypoechogenic at the first days pp and turn into anechoic after the 10th day (p=0.0335), presenting less than 10% debris at this time. Furthermore, a progressive and remarkable reduction of the total uterine diameter were observed (p<0.0001; r2=0.7696), especially during the first days postpartum, and after the 8th day pp it decreased progressively until the end of the evaluations. The measurement of uterine wall thickness gradually decreased (p<0.0001; r2=0.7199) with the advance of days, reducing significantly after 6 days pp. The endometrial thickness progressively decreased (p<0.0001; r2=0.8460) as the days progressed, diminishing after 10 days and becoming almost imperceptible at the ultrasound after 24 days. The myometrium thickness gradually decreased (p<0.0001; r2=0.8471) with the development of days, reducing significantly after 12 days pp, turning into almost undetectable after 20 days postpartum. The diameter of the lumen reduced with the progress of the days (p=0.0002; r2=0.8123), dropping significantly after 8 days p.p. 18 The changes in uterine measurement during the involution process in relation to time are shown in Figure 1. Figure 1. Graphic illustration showing the relation between uterine measurements (uterine wall thickness; biometry of lumen uterine body) and postpartum days in healthy ewes uterus. Regarding to the color Doppler evaluations, the uterus was highly vascularized in all animals, and the flow was easily notable in the first days after parturition (Figure 2A). However, it decreased during the postpartum period, being less evident after the 18th day, and lower (p= 0.0225) on the 30th day (Figure 2B). However, the type of flow, that was mostly mixed and the type of vessel, peripheral, did not present significant variations (p>0.05) during the postpartum period. 19 Figure 2: Ultrasound color Doppler image of uterine blood flow during postpartum in healthy ewes. A: 4 days; B: 18 days; C: 30 days pp. B C A 20 Based on the qualitative elastography of the uterine tissues, all the animals showed that the uterine tissue is hard (dark regions), meaning they were not deformable during uterine involution (Figure 3). In 84% of the elastography assessments presented homogeneous parenchymal echotexture (Figure 3) while 16% heterogeneous echotexture, and there is no relation to the postpartum moment evaluated (p = 0.9010). Figure 3. Ultrasound image of postpartum healthy ewe uterus during its qualitative ARFI elastography analysis. Note the B- mode image (left) and the image of the elastography (right) of the uterus showing a homogeneous and dark (hard) image, respectively. Regarding the quantitative ARFI method, the shear velocities (Figure 4) of the uterine wall did not differ during the postpartum period. The SWV of the uterus and the depth evaluated were constant during postpartum assessment (P = 0.2176 and 0.1027, respectively), and no correlation was observed between SWV and depth of assessment (p = 0.9930) (Table 1). 21 Figure 4. Ultrasound Image of postpartum healthy ewe uterus during its quantitative ARFI elastography analysis. Note the measurement of the shear velocity of uterine wall, with the presence of a caliper for the portion assessed. Table 1. Mean values (±SD) (m/s) for shear waves velocities (SWV) and depth of different moments (days pp) during the postpartum involution in healthy ewes using acoustic radiation force impulse (ARFI) quantitative elastography (Jaboticabal, 2018). Variable SWV Depth Days pp Mean SD Mean SD 0 1.5325 0.3818 3.514 1.2 2 1.4408 0.3386 3.312 1.494 4 1.5613 0.2285 2.887 1.83 6 1.5724 0.3434 2.808 1.479 8 1.58 0.3807 2.838 1.671 10 1.6347 0.3095 2.476 0.828 12 1.4305 0.2099 2.555 0.916 14 1.5293 0.2711 2.896 0.938 16 1.3992 0.2327 3.484 1.097 18 1.5067 0.3085 3.062 0.861 20 1.4291 0.1814 2.727 0.724 22 1.4577 0.1574 2.692 1.274 24 1.3577 0.1843 3.271 1.667 26 1.4375 0.1733 3.136 1.317 28 1.3638 0.16 2.658 1.213 30 1.3593 0.1342 2.958 1.193 22 4. Discussion In this study, we showed the efficacy and the executability of B-mode, color Doppler and ARFI elastography as a safe and non-invasive method for evaluating the assessment of physiological uterine involution during postpartum in sheep. It was established reference data for values of uterine biometry, uterine wall stiffness and blood flow in healthy animals during this period. Shortly after the moment of the partum, there are several physiological uterine modifications for the return of the uterus to enter in a new reproductive cycle (Elmetwally and Bollwein, 2017), such as degeneration of the caruncles, reduction in the size of the uterus and recovery of myometrium and endometrium (Bajcsy, 2005). Initially, the uterine wall is covered with caruncles (Van Wyk et al., 1972a), which could physiologically explain the increased in echogenicity of the uterine wall in B-mode found in this study. In addition, the echogenicity of the uterus may be related to uterine tone and hormonal changes (Viñoles-Gil et al., 2010; Degefa, 2003). Studies reported a difficult to observe caruncles (Ababneh and Degefa, 2005; Kähn, 2004; Fasulkov, 2014; Zongo et al. 2015) and differentiation from uterine layers (Hauser and Bostedt, 2002) by ultrasound around 15 days postpartum, reflecting the images found in this study of hypoechogenicity and isoechogenicity, and no more hyperechoic images after 20 days postpartum. Similarly, Phar and Post (1992) described that during the period of initial uterine involution in bitches, the endometrium showed a hyperechoic characteristic, while the myometrium was hypoechoic and, at the end of this period, Yeager and Concannon (1990) described the uterus as homogeneous and hypoechoic. In felines, myometrium and endometrium were also observed as a hyperechoic characteristic during initial uterine involution, and then the uterine wall became isoechoic compared to adjacent tissues (Ferreti et al., 2000), corroborating the data found in our study with sheep during the same period. A study using B-mode ultrasonography to evaluate uterine regression in ewe, reported that a delay of the separation process of the fetal membranes from the caruncles is characterized by a hyperechoic border of the caruncles with the central site more hypoechoic (Hauser and Bostedt, 2002), supporting our results that the variation in the pattern found in our study may reveal an alteration in the normal course of development of uterine involution. 23 Regarding to B-mode echotexture, a similarity of echotexture in perimetrium and myometrium was observed in ewes (Hauser and Bosted, 2002), as well as similar echotextures of caruncles and the endometrium in goats (Zongo et al., 2015), dificulting differentiation by ultrasonography during the first weeks of postpartum, corroborating to our findings that the parenchymal echotexture did not present significant variations remaining homogeneous during the postpartum period. In the present study, the uterine contents were present and with a hypoechogenic characteristic at the first days pp., corroborating with finds described by Hauser and Bostedt (2002), Ababneh and Degefa et al. (2005) and Badawi et al. (2014), and it decreased gradually by the 22nd day postpartum, when it was no longer observed. This finds corresponds to a study conducted by van Wyk et al. (1972), who found only little amounts of fluid in the uterus up to day 20 pp. The debris observed during the initial days pp. in the uterine lumen may also be tissues, debris and blood normally present within the uterus during the postpartum (Shen et al., 2003). Hauser and Bostedt (2002) suggested that little amounts of fluids seem to be physiological in postpartum uterus. A remarkable reduction in the uterine diameter was observed during the first days pp, however, after this period the reduction was decreased. Similarly, results were reported in ewes by Ioannidi et al. (2017), Elmetwally and Bollwein (2017), Zduńczyk et al. (2004), and Hauser and Bostedt (2002). The ultrasonography revealed to be a useful and reliable method to observe the uterine involution in sheep. Indeed, in our study it was possible to evaluate the measurements of thickness of myometrium, endometrium and the biometry of uterine body. The thickness of uterine wall, myometrium and endometrium decreased with the advance of days, which is may related to the uterine contractility during the early postpartum period, and the regeneration of the uterine layers (Ioannidi et al., 2016). The uterine wall thickness gradually decreased, and reduced significantly at the 6th day pp., related to data reported by Fasulkov (2012), which significant differences in the uterine wall thickness was observed by the 9th day postpartum in goats. The present study presents reference data on the changes in uterine blood flow during the postpartum period in healthy ewes, assessed by color Doppler ultrasonography during the uterine involution process in this period. Our results agreed 24 with findings in ewes, whereupon the uterine blood flow decreased during the postpartum period (Elmetwally and Bollwein, 2017; Elmetwally et al., 2016). This reduction has been associated with the great decrease in the uterine size after parturition, and may be related to the important uterine blood flow changes right after the partum, due to reduction of the metabolic requirements for pregnancy (Elmetwally and Bollwein, 2017). Similar results were reported in cows (Heppelmann et al., 2013), mares (Lemes et al., 2016), as well as in women during the initial postpartum (Van Schoubroeck et al., 2004). According to our knowledge, studies reporting the use of elastography ARFI for evaluation of changes in uterine stiffness during postpartum involution have not been reported in animals. However, a study conducted by Tanaka et al. (2011) in humans evaluated the stiffness of the uterus and cervix before, immediately after, and 1 and 2 h after placental delivery, while our study assessed the uterus during 30 days. Additionally, the great repeatability of the procedure, every 48h, has been demonstrate, highlighting the importance and the consistence of the presented data in this study, as well as proves that the elastography ARFI does not have showed any issues for the animal health. In this study, the uterine parenchyma exhibited as rigid (dark) and homogeneous tissue indicating that was not easily deformable. This is consistent with data of a recent study conducted by Frank et al. (2016) in the evaluation of normal uterine tissue in non-pregnant humans, emphasizing that normal uterine tissue is relatively homogeneous in elastographic evaluation. From the uterine characterization in the physiological puerperium, it reinforces the suggestion that a different elastographic pattern is potentially indicative of alterations, pathological or not, in the uterine parenchyma. Shear wave elastography is based on the concept that shear waves move faster through more rigid regions in a tissue. It has been used successfully in placenta of normal and pre-eclamptic pregnancies in women (Cimsit et al., 2015; Kiliç et al., 2015; Wu et al., 2016) and in the evaluation of cervix in pregnant women (Gennisson et al., 2011; Carlson et al., 2014; Hernandez-Andrade et al., 2014). Additionally, SWV was considered as valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success in 25 pregnant sheep, as animal model (Peralta et al., 2015). In our study, similarly to the qualitative results that demonstrated a consistent pattern of uterine wall stiffness, the quantitative data (SWV) were also constant in relation to the elasticity of the uterine wall in the involution process. These quantitative values obtained by ARFI of uterus in healthy animals are, to the authors’ knowledge, the first that have been described in veterinary medicine. One published study using ARFI (Tanaka et al., 2011), in attempt to quantify endometrium and myometrium in healthy humans, described difference between the means of the SWV in layers, but the authors reported data as not representative to set a reference range. In veterinary research, as mentioned previously, recent studies have described elastography ARFI with significant results, but this is the first study to evaluate the uterine wall parenchyma in animals. The results of the present study suggest that, even though during the postpartum involution the size of the uterus change over the time (Van Wyk et al., 1972b), the elasticity of the uterus undergoes little changes, not being significant. We believe that our study provides important information about the validation of the ARFI technique, and the stiffness of the normal uterus may be useful as a reference parameter for the assessment of uterine integrity during postpartum period. 5. Conclusions The data presented in this study emphasize that ultrasonography is promissory tool to distinguish the pathological from the normal puerperium and thereby avoid unnecessary invasive procedures. It was was possible to describe the physiological changes throughout uterine involution, evaluating the uterine regression development and tissue stiffness during postpartum involution by B mode ultrasonography, color Doppler and ARFI elastography. We provided valuable information to elucidate the mechanism of physiological uterine involution and uterine characterization during the postpartum period in ewe, in order to identify and diagnose potential causes delaying involution. This can be of particular importance to apply in intensive reproductive management, where conception of females is essential, helping in reproductive performance and consequently an economic gain. It is also important to mention that 26 the knowledge of the physiological course of uterine regression is a pre-requisite to diagnose pathologies in practice Acknowledgments To Sao Paulo Research Foundation (FAPESP – Grant 2015/18519-8) and National Council for Scientific and Technological Development (CNPq – Grant 441492/2014-2) for financial support. 6. References Ababneh MM, Degefa T (2005) Ultrasonic assessment of puerperal uterine involution in Balady goats. Journal of Veterinary Medicine Series A, 52, 244-248. doi: 10.1111/j.1439-0442.2005.00718.x Bacjsy AC, Szenci O, Doornebal A, Weijden G, Csorba C, Kocsis L, Szucs I, Ostgard S, Taverne MAM (2005) Characteristics of bovine early puerperal uterine contractibility recorded under farm. Theriogenology, 64, 99-111. doi: 10.1016/j.theriogenology.2004.11.005 Badawi ME, Makawi SEA., Abdelghafar RM, Ibrahim MT (2014) Assessment of postpartum uterine involution and progesterone profile in Nubian goats (Capra hircus). Journal of Advanced Veterinary and Animal Research, 1, 36-41. doi: http://dx.doi.org/10.5455/javar.2014.a10 Carlson LC, Feltovich H, Palmeri ML, Dahl JJ, Munoz Del Rio A, Hall TJ (2014). Shear wave speed estimation in the human uterine cervix. Ultrasound Obstet Gynecol, 43, 452–8. doi: 10.1002/uog.12555 Cimsit C, Yoldemir T, Akpinar IN (2015) Shear wave elastography in placental dysfunction: comparison of elasticity values in normal and preeclamptic pregnancies in the second trimester. Journal of Ultrasound in Medicine, 34, 151–9. doi: 10.7863/ultra.34.1.151 Degefa T. (2003). Postpartum uterine involution in goat in Jordan. MS Thesis. Jordan University of Science and Technology, Irbid, Jordan. Elmetwally M, Rohn K, Meinecke-Tillmann S (2016) Noninvasive colour Doppler sonography of uterine blood flow throughout pregnancy in sheep and goats. Theriogenology, 85, 1–10. doi: 10.1016/j.theriogenology.2015.11.018 Elmetwally M, Bollwein H (2017) Uterine blood flow in sheep and goats during the peri- parturient period assessed by transrectal Doppler sonography. 2017. Anim. Reprod. Sci., 176, 32-39 27 Fasulkov I. (2014). Ultrasonography of uterine involution in Goats. J. Fac. Vet. Med. Istanbul Univ, 40, 63-69. doi: http://dx.doi.org/10.16988/iuvfd.72169 Ferretti LM, Newell SM, Graham JP, Roberts GD (2000) Radiographic and ultrasonographic evaluation of the normal feline postpartum uterus. Veterinary Radiology & Ultrasound, 41, 287–291. doi: https://doi.org/10.1111/j.1740- 8261.2000.tb01493.x Frank ML, Schafer SD, Mollers M, Falkenberg MK, Braun J, Möllmann U, Strube F, Fruscalzo A, Amler S, Klockenbusch W, Schmitz R (2016) Importance of transvaginal elastography in the diagnosis of uterine fibroids and adenomyosis. Ultraschall Med, 37, 373–378. doi: 10.1055/s-0035-1553266 Gennisson JL, Ami OMM, Kohl V, Gabor P, Musset D, Tanter M (2011) Shear wave elastography in obstetrics: Quantification of cervix elasticity and uterine contraction. Ultrasonics Symposium IEEE International. 2094–7. doi: 10.1109/ULTSYM.2011.0519 Hauser B, Bostedt H (2002) Ultrasonographic observations of the uterine regression in the ewe under different obstetrical conditions. Journal of Veterinary Medicine Series A, 49, 511-516. https://doi.org/10.1046/j.1439-0442.2002.00496.x Hayder M, Ali A (2008) Factors affecting the postpartum uterine involution and luteal function of sheep in the subtropics. Small Ruminant Research, 79, 174-178. doi: https://doi.org/10.1016/j.smallrumres.2008.07.023 Hernandez-Andrade, E., Aurioles-Garibay, A., Garcia, M., Korzeniewski, S. J., Schwartz, A. G., Ahn, H., Martinez-Varea, A., Yeo L., Chaiworapongsa, T., Hassan, S. S., Romero R. (2014). Effect of depth on shear-wave elastography estimated in the internal and external cervical os during pregnancy. Journal of Perinatal Medicine, 42, 549–557. doi: 10.1515/jpm-2014-0073 Hernandez‐Andrade E, Hassan SS, Ahn H, Korzeniewski SJ, Yeo L, Chaiworapongsa, T, Romero R (2013) Evaluation of cervical stiffness during pregnancy using semiquantitative ultrasound elastography. Ultrasound in Obstetrics & Gynecology, 41, 152–161. doi: 10.1002/uog.12344 Hwang HS, Sohn IS, Kwon HS (2013) Imaging Analysis of Cervical Elastography for Prediction of Successful Induction of Labor at Term. Journal of Ultrasound in Medicine, 32,937–946. doi: 10.7863/ultra.32.6.937 Ioannidi KS, Mavrogianni VS, Valasi I, Barbagianni MS, Vasileiou NGC, Amiridis GS, Fthenakis GC, Orfanou DC (2017) Ultrasonographic examination of the uterus of ewes during the post-partum period. Small Rumin. Res., 152, 74-85. Jefferies BC (1961). Body condition scoring and its use in management. Taunanian lournal of AgriuúttLre, 32, 79-27 28 Kähn W (2004) Ultrasonography in sheep and goats. In: Veterinary Reproductive Ultrasonography. (Kähn, W., Ed.). Schlütersche Verlagsgesellschaft mbH & Co. KG, Hannover. 187-195. Karaman E, Arslan H, Çetin O, Şahin HG, Bora A, Yavuz A, Elasan S, Akbudak İ (2016) Comparison of placental elasticity in normal and pre‐eclamptic pregnant women by acoustic radiation force impulse elastosonography. Journal of Obstetrics and Gynaecology Research, 42, 1464–1470. doi: 10.1111/jog.13078 Kiliç F, Kayadibi Y, Yüksel MA, Adaletli İ, Ustabaşıoğlu FE, Öncül M, Madazlı R, Yılmaz MH, Mihmanlı İ, Kantarcı F (2015) Shear wave elastography of placenta: in vivo quantitation of placental elasticity in preeclampsia. Diagn Interv Radiol, 21, 202–7. doi: 10.5152/dir.2014 Kiracofe GH (1981) Uterine involution: Its role in regulating postpartum intervals. J. Anim. Sci, 52, 16 (Abstr.). Medan MS, El-Daek T (2015) Uterine involution and progesterone level during the postpartum period in Barbary ewes in north Libya. Open Veterinary Journal, 5, 18- 22. Nasciutti NR, Oliveira RSBR, Silva NC, Franco MT, Tsuruta SA, Ferreira IC (2001) The clinical evaluation of uterine involution in santa inêes sheep. Biosci. J, 27, 649-655. Noakes DE (2001) Part Two: Pregnancy and Parturition; Chapter 2. Development of the Conceptus. In: Noakes DE, Parkinson TJ, England GCW (eds): Arthur’s Veterinary Reproduction and Obstetrics. 8th. ed. London: WB Saunders, 57-68. Peralta L, Mourier E, Richard C, Charpigny G, Larcher T, Aït-Belkacem D (2015) In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography, Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model. PLoS ONE, 10, e0133377. doi:10.1371/journal.pone.0133377 Pharr JW, Post K (1992) Ultrasonography and radiography of canine postpartum uterus. Veterinary Radiology & Ultrasound, 33, 35-40. doi: https://doi.org/10.1111/j.1740-8261.1992.tb01954.x Sanchez MA, Garcia P, Menendez S, Sanchez B, Gonzalez M, Flores JM (2002) Fibroblastic growth factor receptor (FGF-R) expression during uterine involution in goat. Animal Reproduction Science, 69, 25-35. doi: https://doi.org/10.1016/S0378- 4320(01)00169-5 Sheldon IM (2004) The postpartum uterus. Veterinary Clinics of North America: Food Animal Practice, 20, 569-591. doi: 10.1016/j.cvfa.2004.06.008 Sheldon IM, Owens SE (2017) Postpartum uterine infection and endometritis in dairy cattle. Animal Reproduction, 14, 622-629. doi: 10.21451/1984-3143-AR1006 29 Sheldon IM, Williams EJ, Miller ANA, Nash DM, Herath S (2008) Uterine diseases in cattle after parturition. Veterinary Journal, 176, 115-121. doi: 10.1016/j.tvjl.2007.12.031. Shen O, Rabinowitz R, Eisenberg VH, Samueloff A (2003) Transabdominal sonography before uterine exploration as a predictor of retained placental fragments. J. Ultras. Med. 22, 561-564. Tan S, Ozcan MF, Tezcan F, Balci S, Karaoğlanoğlu M, Huddam B, Arslan H (2013). Real-time elastography for distinguishing angiomyolipoma from renal cell carcinoma: preliminary observations. American Journal of Roentgenology, 2000, 369–375. doi: 10.2214/AJR.12.9139 Tanaka T, Makino S, Saito T, Yorifuji T, Koshiishi T, Tanaka S, et al (2011) Attempt to quantify the uterine involution using Acoustic Radiation Force Impulse (ARFI) before and after placental delivery. Journal of Medical Ultrasonics, 38, 21–25. doi: 10.1007/s10396-010-0292-5 Van Wyk LC, Van Niekerk CH, Belonje PC (1972a). Involution of the post partum uterus of the ewe. Journal of the South African Veterinary Association, 43, 19–26. Van Wyk LC, Van Niekerk CH, Bolonje P.C. (1972b). Further observations on the involution of the post partum uterus of the ewe. Journal of the South African Veterinary Association, 43, 29–33. Viñoles-Gil C, Gonzales-Bulnes A, Martin GB, Zlatar FS, Sale S (2010) Sheep and goats. In: DesCoteaux, L., Colloton, J., Gnemmi, G.(Eds.), Ruminant and Camelid Reproductive Ultrasonography. Willey-Blackwell, Hong Kong, 181-210. Wu S, Nan R, Li Y, Cui X, Liang X, Zhao Y (2016). Measurement of elasticity of normal placenta using the Virtual Touch quantification technique. Ultrasonography, 35, 253- 257. doi: 10.14366/usg.16002 Yeager AE, Concannon PW (1990) Serial ultrasonographic appearence of postpartum uterine involution in Beagle dogs. Theriogenology, 34, 523-535. doi: https://doi.org/10.1016/0093-691X(90)90009-I Zongo M, Traoré B, Ababneh MM, Hanzen C, Sawadogo L (2015) Ultrasonographic assessment of uterine involution and ovarian activity in West Africa Sahelian goats. Journal of Veterinary Medicine and Animal Health, 7, 71-76. doi: 10.5897/JVMAH2014.0322 30 CHAPTER 3 - POSTPARTUM EVALUATION IN EWE: STUDY OF UTERINE AND CERVICAL INVOLUTION BY HYSTEROSCOPY AND HISTOLOGY ABSTRACT – To evaluate the time of cervical closure during postpartum in healthy animals, and determine morphophysiological characteristics of this structure observed through hysteroscopy and histology of the uterus and cervix during this period, twenty adult multiparous Santa Ines ewes were selected. Hysteroscopy evaluation of the uterine involution of the sheep was performed as follows: immediate moment after lambing (M0) and sequentially every 6 hours, until the moment where the endoscopic access to the uterus through the cervix was no longer possible. Uterine biopsy was performed at the same moments. Regarding the time of cervical involution, 30% of the animals had a cervical closure between 12-18 hours. The most frequent external os type was papilla 7/20 (35%), followed by flap 5/20 (25%). Histological evaluations showed changes during the postpartum evaluation. This is the first study to evaluate the uterine involution through hysteroscopy in healthy ewes, and may be a useful tool for the early diagnosis of uterine alterations during the postpartum period, avoiding the use of invasive procedures to the assessment of this period. Keywords: cervix, puerperium, sheep, endoscopy. 31 1. Introduction Uterine involution and postpartum fertility may apply limitations on female reproductive performance and fertility (Greyling and van Niekerk, 1991; Greyling, 2000; Ababneh e Degefa, 2005) mainly under intensive accelerated production systems, and this is economically important in small ruminants. Therefore, little has been known about the time, pattern of cervical closure and the macroscopic and histological carachteristics of this physiologic process during postpartum. Uterine cervix is firm and closed during the pregnancy due to a high content of connective tissue, which is surrounded by bundles of smooth muscle cells (Myers et al., 2015). Shortly before and during parturition, there is a remodelletion of the connective tissue of the cervix (Winkler et al., 2003; Engelen et al., 2007). The cervix is an important barrier against the invasion of bacteria in the uterine cavity (Bekana et al., 1997), and the closure of the cervical canal after parturition is important for a successful new pregnancy. When this process is incomplete or delayed, as is the case of retention of the placenta, it predisposes to the development of other pathological processes such metritis, endometritis, and it can leads to lower conception rates, increased of partums intervals and also infertility (Engelen et al., 2007). Endoscopy is a non-invasive tool widely used in reproduction in humans as a diagnostic and treatment technique, since it allows the direct observation and evaluation of the vaginal cavity and related structures (Sardo et al., 2016). However, applications in veterinary medice have not yet been so extensively established, but it has greatest potential that veterinary medicine will likely parallel the trends in human medicine (Katic and Dupre, 2016). In veterinary medicine, several applications for vaginoscopy or histeroscopy have been described in cows (Franz, 2008; Madoz et al., 2010), ewes (Easley et al., 2017), mares (Ferrer et al., 2012) and bitches (Lévy, 2016), with the purpose of diagnosis and small procedures as well as providing an opportunity to take visually guided biopsies, proving to be highly effective (Easley et al., 2017). Vaginal examination also allows to detect damage to the wall of the vagina and cervix, indicative of obstetric injuries, vaginitis, and cervicitis (Sheldon et al., 2018). The purpose of the present study was to evaluate the time of cervical closure during postpartum in healthy animals and to determine characteristics 32 morphophysiological of this structure observed through hysteroscopy and histology of the uterus and cervix during this period in sheep. 2. Material and Methods Ethical aspects All animal procedures were approved by the Animal Ethics and Welfare Committee of the Faculty of Agricultural and Veterinary Sciences, Univ. Estadual Paulista (Unesp), Jaboticabal, Brazil (protocol N ° 12338/15). Animals Twenty adult multiparous healthy Santa Ines ewes, aged 3.1 ± 1.1 years, weighing 45.4 ± 4.3 kg and exhibiting a mean body score of 3 (scale 1-5, Jefferies 1961), were selected for this study following clinical background evaluation, physical examinations, hematological tests and ultrasonography of the reproductive system. Ewes were maintained in an elevated sheep house at the Animal Reproduction Department, fed with corn silage, balanced commercial concentrate, mineral salt and water ad libitum during pregnancy and throughout the postpartum period. Only the animals that had non-intercurrent full-term normal delivery were used in this study. Experimental Protocol The animals were maintained in quadrupedal station, and no sedation was required throughout the entire duration of the exam. The postpartum evaluation of the uterine involution of the sheep was performed as follows: immediate moment after lambing (M0) and sequentially every 6 hours, until the moment where the endoscopic access to the uterus through the cervix was no longer possible. A vaginal speculum was lubricated and inserted into the vagina for the identification of the uterine cervix. Then, it was grasped and pulled with an atraumatic Babcok grasper to visualize the external orifice of the uterus cervix. Through the speculum, a 4 mm diameter, 0∘, 30 cm rigid laparoscopic (Scope electronic optical, GDI Brazil) connected to a canula for endoscopy 5.5 mm (GDI Brazil) was inserted via the cervix in attempt of passage and characterization of the uterus and cervical orifice. 33 Upon entering the vagina, visible structures were identified and classified the following parameters according to Kershaw et al. and Grunert et al. (2005): format of the cervical extern os (duckbill, slit, rose, papilla, flap), mucosal staining, cervical canal opening (1- 5), vaginal and cervical moisture (1-dry;2-slightly moist;3-medium;4-very moist;5- collection of mucus). The examination was video recorded and possible intercurrences and particularities were noted. Uterine biopsy was performed videoassisted with a 2.2 mm laparoscopic flexible biopsy forceps (Huger®, China). When the passage of the optic through the cervix was possible, uterine fragment was collected of the uterine wall, tracing it to its detachment through the working channel of the cannula endoscope. Histology Once collected, uterine fragments were fixed in 10% formalin solution, buffered with phosphates (0.15 Molar), pH 7.2, for 48 hours. Subsequently the tissues were dehydrated in solutions of increasing concentration of alcohol, diaphanized in xylol, and included in paraffin according to the routine histological technique. For the preparation of the slides the blocks were cut at the thickness of 4μm and stained with Hematoxylin and Eosin and analyzed by light microscopy (Tolosa et al., 2003). Statistical analysis Statistical analysis was performed using the software R, version 3.3.0 (R® foundation for statistical computing, Austria). Hysteroscopy variables were compared between postpartum days by exact-Fisher test, quantitative variables by Friedman test and Dunss post-hoc. Differences were considered significant when P-value < 0.05. 3. Results All animals (n=20) of this study had a normal birth with no obstetrical complications and were evaluated every 6 hours after delivery until the cervical closure, established as the moment when it was inaccessible the passage of the optic through the cervix, trying to avoid iatrogenic lesions. If the cannula did not pass easily through the cervix on the first attempt, the cannula was re-positioned and further attempts made. In two animals, the access to 34 the cervix and uterus was not possible. The vaginal wall and cervical canal were easily visualized. However, it was not possible in all the animals and moments collect the uterine tissue through the biopsy. There were no complications due to the examination, but one animal evaluated in this study had uterine prolapse on day 5 postpartum. For the animals evaluated, 25% had cervical closure between 6-12 hours, 30% of 12-18 hours, 25% of 18-24 hours and 10% of 24-30 hours. In 10% of the animals, it was not possible to evaluate the cervical closure (Table 1). Table 1. Correlation of number and percentage (%) of animals evaluated and time of cervical closure observed trough endoscopy during the postpartum involution in healthy ewes. Jaboticabal, 2018. Cervical closure time Number of animals Percentage 6h-12h 5 25% 12h-18h 6 30% 18h-24h 5 25% 24h-30h 2 10% No access to the uterus 2 10% The external os was classified according to the shape, according to Kershaw et al. (2005). The most frequent external os type was papilla 7/20 (35%), followed by flap 5/20 (25%). The remaining types, duckbill (3/20; 15%), slit (3/20; 15%) and rose (2/20; 10%) were the least common. A small lesion in the external cervical os was observed in 3 animals. The mean of the cervical canal open was 2.61±1. The staining mucosal of the animals, showed a normal to hyperemic mucosal, mainly bright pink equally saturated. The mean of the moisture was 2.55±1 (vaginal), and 3.27±1 (cervical). Regarding the histology, 15 samples of uterine tissue were obtained from nine animals, and it was processed and evaluated. The histological findings were compatible with uterine and cervical tissue. The histoarchitectural characteristics observed in the animals were: marked hyperemia of the submucosa vessels, Inflammatory cell infiltration mostly constituted by neutrophils, and apoptosis of the 35 epithelial cells of the endometrial glands. The histoarchitectural pattern found in this study is shown in Figure 1. Figure 1 – Photomicrographs of the histological evaluation of ewes uterus in the postpartum period. A) Cervical region showing moderate hyperemia of the vessels in the submucosal (arrows) and discrete neutrophilic inflammatory infiltrate (Bar = 50μm). B) Uterine epithelium showing increased vascularization (arrow) and marked neutrophilic inflammatory infiltrate in the endometrium (*) (Bar = 50μm). C) Endometrial region presenting the predominance of neutrophils in the inflammatory infiltrate (arrow) (Bar = 50μm). D) Endometrial glands with epithelial cells in apoptosis (*) (Bar = 100μm). Hematoxylin-Eosin. 4. Discussion Various techniques have been proposed for the study of uterine involution in ewes, and most of the disadvantages of these techniques include invasiveness, reduced accuracy, difficulty to apply in clinical conditions and need to sacrifice the 36 experimental animals. In our study, the hysteroscopy in ewes has shown to be a noninvasive procedure, and atraumatic during normal, gently and proficient procedures. It was not possible in all animals and moments to collect the uterine tissue through the biopsy, due to the anatomical conformation of the sheep cervix, which is highly variable between animals, and may explain the difficult to access the uterus (Kershaw et al., 2005), as well as the behavior of some animals postpartum made the procedure difficult. Similar limitations were recorded in a previous study in bubalines, when the circular annular rings in the cervix presented resistance to the passage of the hysteroscope (Chaudhary et al., 2014). We believe the placenta present during hysteroscopic evaluations, as well as large amounts of fluid through the cervix in the postpartum immediate moment (M0), made difficult the passage of the endoscopic through the cervix and the visualization to the uterus, and affect the success rates in the procedure. Madoz et. Al. (2010) describe the use of a disposable plastic sleeve to cover the endoscopy during postpartum hysteroscopy. Moreover, the rigid endoscope does not enable to gain access and passage through the cervical rings with no insufflation, when cervix appeared closed, as same as observed in a study conducted by Watts and Wright (1995) to investigate uterine diseases in bitches. However, same authors have reported uterine tear during postpartum hysteroscopy due to the pressure of the insufflation, because after parturition the uterine wall may be weakened. Even though some authors have described the use of a rigid endoscopy to examination of the uterus in cows (Madoz et al., 2010) and buffalos (Chaudhary al., 2013), they also reported it does not allow exploration of the whole uterus, mainly endometrium, and limits exploration. The results of this study indicate the most frequent external os type in Santa Ines sheep was papilla followed by flap, similarly the most common finds in ewes reported by Halbert et al. (1990) and Kershaw et al. (2005) in cervices evaluated after slaughter. Dun (1955) suggested that the classification of the cervical os might change at parturition, increasing in size and complexity. The opening of the cervix favors the entry of bacteria leading to an immediate cellular immune response (Sheldon, 2004), and besides the physiological infection 37 developed, the uterus starts a regenerative process (O'shea and Wright, 1984), which may explain the inflammatory cell infiltration observed in the histological analyses in our study. Gray et al. (2003) reported a significant increase of neutrophils and macrophages from the first to seventh postpartum days in ewes. Therefore, it suggests that that increase it is physiological according to the changes of the intrauterine environment, being directly associated with phagocytosis of placental structures remaining from postpartum (Nasar et al., 2002; Gray et al., 2013). In addition to these changes, Gray et al. (2013) described a marked reduction in vascularity in both caruncular and intercaruncular areas of the uterine wall during involution, controverting our finds, whereupon the uterine epithelium showing increased vascularization. This results may be explained by the increase of the uterine blood flow observed through color Doppler in ewes during postpartum (Elmetwally et al., 2016). 5. Conclusion These data provide the first evidence that hysteroscopic examination of the uterus in ewes can be used to determine the progress of the involution, and may reveal in evidence details in which cannot be assessed through rectal palpation, and with more detail than obtained by ultrasonography. The technique we have described for evaluate of the uterine involution will be a valuable procedure which will assist in early diagnosis and treatment of uterine disease in ewes, thus avoiding invasive procedures. For future studies, the technique could be improved. Acknowledgement To Sao Paulo Research Foundation (FAPESP – Grant 2015/18519-8) and National Council for Scientific and Technological Development (CNPq – Grant 441492/2014-2) for financial support. 6. References Ababneh MM, Degefa T (2005) Ultrasonic assessment of puerperal uterine involution in Balady goats. J. Vet. Med. A Physiol. Pathol. Clin. Med. (52):244-248 38 Basarab T, Stefanyk V (2016) Hysteroscopic Investigation Of Dairy Cows Uterus With Subclinical Endometritis. Scientific Messenger of Lnu of Veterinary Medicine and Biotechnologies 18:3(71). Cicinelli E (2010) Hysteroscopy without anesthesia: review of recent literature. J Minim Invasive Gynecol 17(6):703-8. Chaudhary V, Jeengar K, Ruhil S, Purohit GN (20130) Efficiency of hysteroscopic visualization of bubaline uterus. Animal Reproduction Science , Volume 149 , Issue 3 , 353 – 355. Dun R. (1955) The cervix of the ewe. Its importance in artificial insemination of sheep. Aust.Vet. J.,101–103. Elmetwally M, Rohn K, Meinecke-Tillmann S. (2016). Noninvasive colour Doppler sonography of uterine blood flow throughout pregnancy in sheep and goats. Theriogenology, 85, 1–10. doi: 10.1016/j.theriogenology.2015.11.018. Franz S. (2008) Importance of endoscopic techniques for diagnosis and therapy in ruminants. In XXV Jubilee World Buiatrics Congress. Budapest, July 6 to 11, 2008. pp 33-35. Engelen MA, Taverne ME, Everts GC, Van der Eijden A, Doornenbal VN. Breeveld Dwarkasing (2007) Cervical diameter in relation to uterine and cervical EMG activity in early postpartum dairy cows with retained placentas after PGF2alpha induced calving. Theriogenology, 68, 213-222. Gray CA, Stewart MD, Johnson GA, Spencer TE (2003) Postpartum uterine involution in sheep: histoarchitecture and changes in endometrial gene expression. Reproduction, v.125, 185-198. Greyling JPC, Van Niekerk CH (1991) Macroscopic uterine involution in the post- partum Boer goat. Small Rumin. Res. 4, 277-283. Greyling JPC (2000) Reproduction traits in the Boer goat doe. Small Ruminant Research, 36 (2)171–177. Halbert GW, Dobson H, Walton JS (1990) Field evaluation of a technique for transcervical intrauterine insemination of ewes. Theriogenology, v. 33, n. 6, p. 1231- 1243. Easley J, Shasa D, Hackett E (2017) Vaginoscopy in Ewes Utilizing a Laparoscopic Surgical Port Device. Journal of Veterinary Medicine, vol. 2017, Article ID 7404371, 4 pages. https://doi.org/10.1155/2017/7404371. Jefferies BC (1961) Body condition scoring and its use in management. Tasmanian J Agr, 32, 19–21 Kershaw MC, Khalid M, McGowan MR, et al (2005) The anatomy of the sheep cervix 39 and its inluence on the transcervical passage of an inseminating pipette into the uterine lumen.Theriogenology. 64: 1225-1235. Lévy X (2016) Videovaginoscopy of the canine vagina. Reproduction in Domestic Animals. 51, 1:31–36. doi: 10.1111/rda.12785. Madoz, L, Luzbel R, Suzuki K (2010) Use of hysteroscopy for the diagnosis of postpartum clinical endometritis in dairy cows. The Veterinary record. 167. 142-3. Myers KM, Feltovich H, Mazza E, et al (2015) The mechanical role of the cervix in pregnancy. Journal of biomechanics. 48(9):1511-1523. doi:10.1016/j.jbiomech.2015.02.065. Nasar A, Rahman A, Meeusen ENT, Lee CS (2002) Peri-partum changes in the intraepithelial lymphocyte population of sheep interplacentomal endometrium. American Journal of Reproduction and Immunology, v, 47, p. 132-141. O’shea, JD, Wright PJ (1984) Involution and regeneration of the endometrium following parturition in the ewe. Cell and Tissue Research, v. 236, p. 477-485. Sheldon, IM. (2018) Metabolic stress and endometritis in dairy cattle Veterinary Record 183, 124-125. Watts JR, Wright PJ (1995) Investigating uterine disease in the bitch: Uterine cannulation for cytology, microbiology and hysteroscopy. Journal of Small Animal Practice, 36, 201–206.