Cytological Characteristics of Mucose Cell and Vaginal Temperature and PH During Estrous Cycle in Local Sheep

Aim of this study was to examine the characteristics cytology of mucous cell-,temperature- and pH vagina during estrous cycle in local sheep. 31local sheep were synchronized with vaginal sponge consist of 20 mg progesterone hormone before carried out observations of cytology of cells from the vaginal mucose through vaginal swabs, temperature and pH of the vagina. Vaginal swabs were collected daily at 7 am for a weeks.Vaginal temperature and pH measurement is carried out twice a day, at 07.00 am and 15.00 pm for a weeks after vaginal swabs. Smears of the swab were then prepared on glass slide and they were stained with Giemsa. Vaginal epithelial cells; Parabasal, intermediate and superficial cells were counted and their percentages during pro-estrous, estrous and di-estrous were determined. Di-estrous was characterized by the absent of superficial cells in the epithelial vagina. Pro-estrous was characterized by the increasing progressively of intermediate/superficial cells in epithelial vagina, whereas estrous was characterized by the presence of superficial/cornification cells in most epithelial vagina. Based on the dominance of superficial cell, the number of sheep identified as estrous is highest on third day, with 52%. Observation on vaginal temperature also resulting that the highest temperature values obtained on the third day of 39,08±0.28°C. It could be effected of the vaginal pH during the observation. Underthe influence ofestrogen, the epithelial vaginalcellssynthesizeand accumulateglycogenin large quantitiesdepositedin the lumen ofvagina. Vaginal bacteriametabolizethe glycogenformlactic acid, which causesvaginal pHis low.The pH conditions prevent from pathogenic microorganisms and fungi. Increased estrogenal so cause cell proliferation through the thickening of the epithelium lining of the vagina so that the cells differentiate.Increasing of glycogenin the superficial cells, and ceratin cells found in the cytoplasm of cells, as well as acidic pH in the lumen vagina causing changes in the chemical composition of each cell (Zaid, 2011).It can be concluded that during estrous phase, cytology of vaginal mucose cell is dominated by superficial cell and vaginal temperature is increasing as well as pH of vagina.


Introduction
Behavioral estrous in livestock animal like sheep or cattle can be detected from the exterior behavior or changes in vaginal cytology during estrous cycle. During ovulation, there are changes in ratio of cornification of the epithelial cells and basal cell (Zaid, 2011), and change in behaviour, body temperature and physical properties of genitals fluids of female cattle due to reproductive hormone status during estrous cycle (Redden et al., 1993). Evaluation of estradiol levels, vaginal cytology, and the appearance of the vulva can be used as a predictor of the estrous cycle in female (Mayor et al., 2006). During estrous, sheep will be affected by estrogen and the symptom of estrous will appear visually from the behavior or changes in the vulva of the sheep. However, sheep often experience reproductive disorder like silent estrous which is a phenomenon of ovulation without signs of estrous, especially common in young sheep. Silent estrus often occurs during the first estrus cycle, which may also be due to estrogen deficiency. In these conditions, the female sheep will not show the symptoms of estrus so it cannot be detected visually. Determination of this estrus cycle in addition to visual observations can be done by the vaginal smear method. Through this method the changes of cytology of epithelial cell and polyneclear leucocyte cells in the vaginal mucus can be seen (Vidal et al., 2013).
To date, research on the characteristics of vaginal cytology during estrous cycle in local sheep is still limited. Therefore, the objective of this research is to examine the characteristics cytology of vaginal mucous cell, temperature and pH during estrous cycle in local sheep.

Materials and Methods
The descriptive research used 31 local sheep for synchronization using vaginal sponge impregnated with 20 mg progesterone hormone before vagina swab as the sample. Upon sponge removal, vaginal swab was conducted every day at 7 am for a week. Estrous phase was justified through estrous symptom and cervix dilatation using speculum and then evaluated the condition of the cervix. Vaginal temperature and pH was measured twice a day (at 7 am and 3 pm) for a week using thermometer and pH meter. Smears of the vaginal swab were then prepared on glass slide and stained with Giemsa.
Parameters of the research were (1) cytology cell of mucous vagina.  Table 1. Table 1 explains that vaginal swab on day 1 is dominated by intermediate cell, indicated higher percentage than the other cells. Day 2 shows declining percentage of intermediate, parabasal and ceratine cells, but increasing superficial cell percentage. On day 3, superficial cell percentage was still higher (41.03%) than the other cells, therefore increasing for 2 days straight. According to Widiyono et al. (2011) superficial cell percentage of goat could increase from proestrous to estrous phase and tend to decline after estrous phase because of estradiol change in blood. Day 4 indicates a decline in superficial cell percentage, an increase in while ceratine cell and a stable intermediate cell percentage. On day 5, ceratine cell was high, in contrast with the declining superficial cells, and it prolonged to day 6 and 7. Parabasal cell and intermediate cell began to increase, conversely with superficial and ceratine cell. Changes of cytology cells in vaginal mucosa of local sheep are presented in Figure 1.

The observation of epithelial cells percentage of vaginal mucosa in local sheep consisted of parabasal cell, intermediate cells, superficial cells and ceratine cells is presented in
Cytology changes of vaginal mucosa cell occurred because of changes of hormonal level during estrous activities in ovary. In estrous phase, level estrogen in blood was high, this could be impact to increasing of uterus activities and make hyper secretion, ceratination of epithelial vagina cell and then increased of mitotic cell and proliferation of epithel cell (Popalayah et al., 2013). Ceratination process was important to protect epithelium of vagina from pathogenic invasion between cell or tissues (Hussin et al., 2014). Endogenous bacteria could be metabolized glycogen in cell to lactic acid and could be decline of pH vagina for the protecting of the pathogenic bacteria.
This observation about cytologies cell of mucose vagina was supported through observation of dilatation of cervix. This justification of cervix dilatation was done to observed peak of estrous and the dominance of cell could be detected. Observation of cervix dilatation is shown in Figure 2.  Cervix dilatation is a character of sheep estrous. Based on cervix dilatation observation, the number of female sheep with open cervix or dilated the cervix tend to increase from Day 1 after removal of sponge vaginal to Day 3, then tend to decline until day 7. Cervix dilatation occurred because of relaxation of cervix and normally done when estrous as a effect of increasing of estradiol level in blood, oxytocin receptor (OxR), cyclooxygenase-2 (COX-2), prostaglandin-F2 (PGF2) (Kershaw et al., 2005). Rodriguez-Pinon et al. (2014) mentioned that estradiol and LH will be increased during estrous. In pre ovulation, estrogen could be increased sensitivity of cervix as an effect of oxytocin activities and OxR. Oxytocin could be initiate cervix to produce in vitro PGF2 and could be affected to mediate COX-2 to catalyzed arachidonic acid to the prostaglandin. Prostaglandin activity then could be effect of cervix dilatation.
In Figure 2 showed, that increasing number of sheep (31 sheep) at day 3 (68%) was cervix dilated condition. According to Vivanco (1986), sheep will be estrous 2 days after vaginal sponge removed, and began at day 3 estrous was done. Feradis (2010) mentioned that estrous phase could be done 24-36 h after metestrous phase. Table 1 showed that day 3 almost sheep were in estrous phase based on superficial cell in vaginal mucose cell and began day 4 until day 7, the percentage of lamb in estrous phase tend to decline to anestrous phase (Figure 3).

Peak estrous
Temperature was measured twice a day (in the morning and evening) for a week after removing vaginal sponges, as presented in Table 2.
Based on Table 2, the average temperature of the vagina sheep on the first day was 38.96±0.32℃, but on the second day the temperature was decline. Otherwise, on the third day when the peak of estrous predetermined through cervical dilatation (Figure 3) was high, and the next day the temperature declined. Vaginal temperature on the third day also showed as a higher temperature than the other days.
On day 5, the temperature was high after declined on day 4, increased on day 6 and continued to decrease on day 7. The temperatures would increase at the estrous phase, and decreased again at the time of ovulation, the rebound during the luteal phase (Khalifa et al., 2010).  Figure 4.
Vaginal temperature up to 24h before estrous increased by 0.23°C. Previous study on goats by Popalayah et al. (2013) showed that vaginal temperature during estrous increased by 0.4°C for 6h to 18h and decreased gradually from 24h to 36h before the end of estrous.  (Hardjopranjoto, 1995). High estrogen at the time of estrous due to increasing blood circulation in the vaginal area and increased fluids in cells resulted in swelling vulva and vagina. Swelling in the vulva and vagina is due to the increased blood flow by estrogen activities and its receptor were found in blood vessels and clinically lowering vascular resistance (Heffner and Danny, 2005).

Estrous detection based on pH Vagina
Vaginal pH conditions during observations could be seen in Figures 5. In that graphic could be seen pH changes for a week. Based on Figure  5, the higher pH of vagina was in day 3, and this according to the estrous cycle that occurs where the peak estrous was on day 3 after removed of vaginal sponge. At day 2, pH vaginal tend to increase until day 3, and then continuously decline until day 5. It was happened that at day 6 and day 7 the pH vaginal increased back again until maximum 7 and last day of observation was 6.9, because normally pH vaginal is acidic.
According to animal behavior since estrous phase, normally at that time the female in estrous phase, it was happened increasingly the body temperature as well pH vaginal. That condition closed related with hormonal activities during estrous period.
Based on data during observation, it could be cleared that pH vaginal was influenced of reproduction behavior and physiological changes in the reproductive organs can be seen during estrous cycle which is a manifestation of the increased estrogen in the blood. Toelihere (1993) suggest that elevated levels of estrogen lead into estrous female animals that marked the emergence of symptoms of lust like vulva swollen, red, warm, slimy, and silent when ridden by a male. Effect of estrogen results in changes in the physicochemical properties of the mucus of the cervix so that a change in viscosity (Darodjah, 2002). Decrease in vaginal pH will return coincided with the process of ovulation (Khalifa et al., 2010). Rezac et al. (2001) also reported that at the end of estrous, vaginal mucus is alblaskaline pH. A decrease in vaginal pH depends on the addition of ions in the vagina, such as hydrogen, sodium and chloride, as well as a decrease in pH is also caused by the accumulation of glycogen and protein (Khadiga et al., 2005). Blaszczyk et al. (2004) mentioned that the decrease in pH coincides with the release of LH in ovulation process.

Conclusions
The study conclude that cytology cells from the vaginal mucus of local sheep during the estrous phase were dominated by superficial cells.
During estrous period, vaginal temperature and pH of local sheep increased.