Original Article Bufalo Bulletin (April-June 2020) Vol.39 No.2 DETERMINATION OF EARLY PREGNANCY IN SWAMP BUFFALOES (BUBALUS BUBALIS) USING VAGINAL CYTOLOGY Wilasinee Inyawilert1,*, Yu-Jing Liao2, Pin-Chi Tang3, Kwanruan Junsong1 and Vuttikai Paungsukpaibool4 ABSTRACT using ultrasonography and rectal palpation on 90 days after AI. The results showed that twenty one The accuracy of pregnancy diagnosis is bufaloes were pregnant, and nine bufaloes were important for increasing reproductive efciency not pregnant. Therefore, the vaginal cytology and decreasing interbreeding intervals of swamp technique might be an alternative method to bufaloes. Hence, the aim of this study was to determine pregnancy and reduce estrus detection investigate the early pregnancy diagnosis in swamp problems in swamp bufaloes. bufaloes by the vaginal cytology. Thirty female bufaloes at 4 to 7 years of age were selected and Keywords: Bubalus bubalis, bufaloes, pregnancy, administered Ovsynch (GnRH + PGF2α + GnRH) vaginal cytology, swamp bufaloes and modifed Ovsynch (GnRH + PGF2α protocols. The vaginal swab was collected from each bufalo after 19 to 21 days of artifcial insemination (AI), INTRODUCTION stained with Wright Giemsa, and observed under microscopy. The results showed that superfcial The swamp bufalo (Bubalus bubalis) is an epithelial cells were more dominant when indigenous and durable breed mostly originating rebreeding (non-pregnant) on 20 to 21 days after from northeastern Thailand, (Triwitayakorn et AI. In contrast, a vaginal smear during pregnancy al., 2006). Nowadays, the bufalo population is was characterized by few superfcial epithelial drastically reducing in Thailand. From 1995 to cells while the parabasal cells and leukocytes were 2004, the number of bufaloes has been decreased more dominant in pregnant bufaloes on 19 to 21 from 3.7 to 1.49 million heads on an average decline days after AI. The results showed that eighteen of approximately 22% per year (Triwitayakorn bufaloes were pregnant, and twelve bufaloes were et al., 2006). As a consequence, conservation of not pregnant using vaginal smear technique. The swamp bufaloes and their sustainable utilization accuracy of vaginal smear technique was 85.71%. in the production system is of utmost necessity Moreover, pregnancy diagnosis was confrmed (Bunmee et al., 2018). In this regard, artifcial 1Department of Agricultural Science, Faculty of Agriculture Natural Resources and Environment, Naresuan University, Phitsanulok, Thailand, *E-mail address: [email protected] 2Division of Physiology, Livestock Research Institute, Council of Agriculture Executive Yuan, Tainan, Taiwan 3Department of Animal Science, National Chung Hsing University, Taichung, Taiwan 4Livestock ofce, Nong Khayang, Uthaithani, Thailand 237 Bufalo Bulletin (April-June 2020) Vol.39 No.2 insemination (AI) can play an important role for intermediate cells are bigger than intermediate animal conservation and improvements in the cells and have angular edges. Superfcial cells are genetic capability of the animals. However, various similar in size to superfcial intermediate cells and reproductive disorders in bufalo, such as delayed nuclei are pyknotic and lacking (Ayen et al., 2012). puberty and maturity, poor estrus expression, long In mice, most nucleated epithelial cells present at postpartum anestrus, distinct seasonal reproductive proestrus stage when using vaginal cytology to pattern, prolonged intercalving intervals, and low investigate the stages of estrus cycle. The estrus conception rates particularly when bred artifcially, stage is distinctively characterized by cornifed deeply afect their reproductive efciency (Warriach squamous epithelial cells, which occur in clusters et al., 2015). Therefore, in order to improve the and no visible nuclei. The cell mixture with a reproductive management in livestock, it is critical predominance of leukocytes, a few nucleated to establish a precise method for determination in epithelial, and/or cornifed squamous epithelial the estrus and early pregnancy (Reese et al., 2018). cells show at metestrus stage. The diestrus stage A successful estrus detection technique and predominantly consists of leukocytes (Caligioni, timing, especially before the onset of next estrus 2009a). In cattle, the cell samples are collected by and rebreeding of those animals, can signifcantly a vaginal swab and stained with 10% giemsa to improve proftability from animal farming. Recent detect the estrus stage. The results show that the eforts to overwhelm the problem of weak estrous dominant proportion of superfcial cells exhibit at behavior was assumed to be the cause of a decline in estrus stage. breeding efciency in cattle and bufalo using many In addition, early pregnancy diagnosis is methods such as a vasectomized teaser, plasma and critical management and economic ramifcation milk progesterone, estrus behavior, painting of tail, because it can shorten the calving interval and a pedometer, vaginal cytology, cervical mucus fern rebreed cattle at the earliest opportunity (Balhara pattern test, vaginal temperature measurement, et al., 2013). Nowadays, we apply many techniques ultrasound, and rectal palpation (Singh et al., to detect early pregnancy, such as rectal palpation, 2000; Holmann et al., 1987; Broes and LeBlanc, ultrasound, measurement of progesterone, and 2014; Narendran et al., 1979; Bernardi et al., 2016; glycoprotein (PAG) tests; however, each technique Sakatani et al., 2016; Honparkhe et al., 2004). has its own benefts and limitations (Reese et al., Vaginal cytology is a simple method and has 2018). Rectal palpation is confned even when been done for estrus detection in many animals performed by experience technicians, from including mice, cow, dog, goat and swamp bufalo approximately 45 days onwards (Perera et al., (Suebkhampet and Marcou, 2019; Ayen et al., 2012, 1980). Transrectal ultrasonography is an accurate Sharma and Sharma, 2016). Diferent types of method for pregnancy diagnosis as early as 26 epithelial cells presented in vaginal smear during day post-insemination, but the cost is quiet high the estrus cycle such as parabasal, intermediate, and an appropriate veterinary technicians are and superfcial (cornifed) cells. Parabasal cells are necessary (Kastelic et al., 1991). Occasionally, small-round cells with round nuclei and have the chemical based pregnancy test and the biological highest nucleocytoplasmic ratio. Intermediate cells marker such PAGs can use for pregnancy diagnosis are larger than that of parabasal cells. Superfcial in blood and milk but it is limited in cost (Reese 238 Bufalo Bulletin (April-June 2020) Vol.39 No.2 et al., 2018). Recently, vaginal cytology has been and rolled in the vagina to remove cells. Cells were conducted for estrus detection, but there are no transferred to pre-heated dry glass slide by rolling available reports regarding various types of cells the swab across the slide. The smeared slides were in vaginal smear for pregnancy diagnosis of air-dried in room temperature for 2 minutes and swamp bufaloes. Therefore, in the present study, then stained with Wright-Giemsa stain (Sigma- we attempted to detect the pregnancy of swamp Aldrich, St. Louis, MO, USA) for 2 minutes. The bufaloes using vaginal cytology and provided a slides were rinsed with water, air-dried and viewed reference. immediately at 100 x magnifcation under light microscopy. The vaginal epithelial cells (parabasal, intermediate, superfcial, and leukocyte cell) MATERIALS AND METHODS were observed according to each group to the determined pregnant and non-pregnant swamp Animals and fxed time artifcial insemination bufalo. The parabasal cell is small epithelial cell, Thirty female swamp bufaloes at 4 to having a round shape and presenting high nuclear- 7 years of age were selected and administered cytoplasmic ratio. The intermediate cell has with a modifed Ovsynch protocols. The swamp variation of shape and is 2 to 3 times bigger than bufaloes in Ovsynch (n = 17) group were received parabasal cell. The superfcial epithelial cell was gonadotropin releasing hormone (4 µg of GnRH large and fat but does not always have the pyknotic analogue; Receptal, Intervet; 2.5 mL) on day nucleus. 0, followed a PGF2α analogue (cloprostenol 250 µg; MSD Animal Health; 2 mL) on day 7. Ultrasonography and rectal palpation A second-treatment of GnRH analogue was The pregnancy and non- pregnancy results given 48 h after PGF2α. In addition, the swamp were confrmed using rectal amniotic sac palpation bufaloes in modifed Ovsynch (n = 13) group were on 90 days after AI. Finally pregnancy was administered with GnRH injection on day 0 and confrmed by rectal ultrasound examinations and PGF2α on day 7, respectively. All swamp bufaloes performed using a portable transrectal B-mode were inseminated at two fxed time, 48 h and 72 h ultrasound scanner on 90 days after AI. The after PGF2α. uterine horns were scanned from their dorsal and lateral surfaces. Determination of pregnancy using vaginal cytology Statistical analysis The AI was performed on the female Data were analyzed using a statistical swamp bufaloes at 4 to 7 years of age, and early program (SAS Enterprise Guide 4.1, SAS Institute pregnancy was determined using vaginal smear Inc., Cary, NC, USA). The signifcance of the technique on 19 to 21 days after AI. To collect cells diferences was analyzed by chi-square test, from the vagina, a sterile cotton tipped swab was and P<0.05 was considered to be statistically rinsed with normal saline and was gently