during early pregnancy in buffaloes

Aim: The present work was carried out to study and compare the variation in physical and rheological (study of stretch and elasticity) properties of cervico-vaginal mucus of pregnant and non pregnant animals which could be used for early pregnancy diagnosis in buffaloes. Materials and Methods: Cervico-vaginal mucus samples were obtained from 30 buffaloes (n=12 pregnant, n=18 non pregnant) and a comparison was made between values of different parameters obtained from pregnant and non pregnant animals. Transparency, consistency, and elasticity of mucus were studied during early pregnancy. Results: Transparency and consistency did not show any remarkable difference between pregnant and non pregnant animals during any phase of estrous cycle. However, rheological property i.e. elasticity, showed a significant difference in its value on th th 9 day of estrous cycle. Spinnbarkeit value decreased significantly in pregnant animals on 9 day (3.9±0.45 cm), whereas non th pregnant group did not show a remarkable difference (8.4±0.43). Spinnbarkeit value was unidentifiable from 12 day onward in both the groups. Conclusion: These results conclude that significant variation in mucus elasticity could be helpful in designing user friendly tool for early pregnancy diagnosis in buffalo in future.


Introduction
non pregnant animals which may help in future for development of an early pregnancy diagnosis tool in Cervical mucus (CM) is continuously produced by buffaloes.the secretory cells of the endocervix, and its quality

Materials and Methods
and quantity vary depending upon the hormonal status of the estrous cycle [1].Pregnancy detection in the Ethical approval: Adequate measures were taken to bovine has been the subject of numerous investigations minimize the pain or discomfort in accordance with the aimed at developing more practical and reliable International Animal Ethics Committee.Study was diagnostic methods.Manual palpation via rectum approved by the committee framed for the research by generally has been considered as the standard and the university authority.common method but it cannot be reliably applied Study area: The present study was conducted from before two months of gestation [2].Some researchers December 2011 to March 2012 at Govind Ballabh Pant have claimed that pregnancy determination with rectal University of Agriculture & technology.Cervico-vaginal palpation is risky even if implantation has been mucus samples were obtained from 30 buffaloes (n=12 achieved [3,4].Other methods like radioimmunoassay pregnant, n=18 non pregnant) (Table -1) and a comparison (RIA) for progesterone assay in milk/ plasma and early was made between values of different parameters pregnancy factor (EPF) determination have limited obtained from pregnant and non pregnant animals.applicability under field conditions due to high cost of Color, consistency and elasticity of cervico-vaginal equipment, lack of sophistication as well as non mucus were taken immediately after collection.availability of highly trained technical staff.
Considering the lack of an ideal method of early Selection of animal: Samples of cervico-vaginal pregnancy diagnosis, the present work was undertaken mucus were taken from the cervix of normal buffaloes to study the variation in physical and rheological of 3-6 years age and 2-3 parity, which were free from properties of cervico-vaginal mucus of pregnant and any genital tract infection.irrespective of the stage of their estrous cycle (day of immediately.Then they were stored at -20OC till GnRH treatment, day 0), followed by 2 ml of PGF2á further processing without added preservative.(Cyclix, 263 µg/ ml Cloprostenol Sodium, Intervet Pregnancy was confirmed after 60 days on the basis of India private Limited) day 7, and a second-GnRH per rectal palpation.Pregnancy and non pregnancy treatment (2.5 ml) 48 h after PGF2á (day 9).Out of samples were separated and different samples taken on 100, those 30 animals which exhibited heat within 24-different days were pooled separately.48 hours of treatment were selected for the experiment.
Mucus was studied for transparency and consistency by visual examination.These properties Heat detection: Estrus was detected twice daily, in were identified and classified as per Sukhdeo and Roy morning and evening.The buffaloes were also [5].monitored both for behavioural symptoms (frequent The transparency of cervico-vaginal mucus were micturition, bellowing, raised tail, restlessness and classified into three main types as follows.licking of external genitalia by other animals) and 1. Transparent: like white of an egg physical changes (vulvular edema and mucus 2. Turbid: cloudy in appearance discharge) of the reproductive tract.On the fifth day 3. Dirty: not homogeneous in look, dirty, colors like after estrus, the corpus luteum (CL) was detected by yellow, ash, grey, red etc. were sometimes mixed.per rectal palpation to confirm the estrus phase.
The consistency of cervico-vaginal mucus was Breeding of animal: Buffaloes showing signs of true classified into two main types as follows.estrus were bred 12 hours after the onset of estrus by 1. Thin: which could flow easily on a glass slide kept natural breeding with high quality bull.

Thick: when mucus samples remained sticky on a
Collection of cervico-vaginal mucus glass slide and did not flow at 45Oangle.Collection procedure: Vulva was washed with a warm The elasticity of cervical mucus was observed by dilute solution of savlon.Sterilized vaginal speculum dropping some mucus from catheter to glass slide and was inserted into vagina and fixed.Sterilized long then it was classified as follows.handle forceps holding cervical sponge was gently 1. Less Elastic: Less viscous, sometimes watery and inserted through vaginal speculum via vagina upto the formed no rope when dropped from the catheter to the cervix.Extreme care being used and line of least glass slide.resistance always taken.Sponge was left there for few 2. Elastic: Having the property to form rope or string minutes and then taken out.Absorbed cervical mucus when dropped from the catheter to the glass slide.was collected in Eppendorf tubes.
3. Very Elastic: The secretion was too viscous and was Samples were taken from 30 bred animals at a difficult to drop from the catheter on glass slide and gap of every 2 day, starting from day of breeding to the remained sticky.completion of one estrous cycle i.e.21 days, Length of the rope formed was measured in considering day 0 (day of breeding),3,6,9,12,15,18,21.

palpation.
Generally coloured cervical mucus have been Storage of sample: The samples were collected in reported to be a conducive factor for sperm penetration Eppendorf tubes and immediately transferred to and conception whereas, turbidity in estrual mucus laboratory on ice where they were marked with date, arrests sperm motility [6].The estrual mucus was sample no and time of collection.Transparency, always transparent whereas the mucus, one day after consistency, elasticity and pH of sample were taken 3).This result is in conformity with that of Mehmood et from 3 days after estrus were yellow or white.As al., [12] who also found significant difference in shown in Table-2, mucus was turbid from day 6 to day 9 spinnbarkeit value between pregnant and non pregnant th th and then changed to dirty from 12 day onward in both animals from 9 day onward whereas Rutllant et pregnant and non pregnant group.The cervico-vaginal al.
[13], did not find any significant difference in the mucus of non pregnant animal became turbid again on spinnbarkeit value of cervico-vaginal mucus of st 21 day whereas in case of pregnant animal, it was pregnant and non pregnant animal.found dirty from 18-21 days and was very less in These results are also in accordance with the results of Scott Blair et al., [14] who found that in amount containing cellular debris.There is probability general mucus of pregnant animal was stiff and plastic that cellular debris is the result of breakdown from the rather than elastic whereas mucus of non pregnant epithelium of the reproductive tract [7].The results are animal was more elastic.in general agreement with the findings of Woodman The increased elasticity of mucus also increases and Hammond [8], Sharma et al., [9] who also found the permeability of cervical mucus to spermatozoa.the similar color patterns of cervico-vaginal mucus.As The results are in agreement with those obtained by far as consistency of cervico-vaginal mucus is Marcus and Marcus [15], who have reported that the considered, the results were very obvious.Majority of Spinnbarkeit gradually increases during proliferative fertile estrus had thin consistency.Similar findings of phase of cycle reaching a maximum at the time of cervico-vaginal mucus consistency have been reported ovulation.Spinnbarkeit is attributable to the presence by Rangnekar et al. [10], Rajesh et al. [11] In both of long chain of molecules in the mucus and is probably pregnant and non pregnant animal, mucus was very rd also dependent upon branching of molecular chains thin during estrus but after 3 day onward the and other strong intermolecular forces Modi et al., [16].consistency became thick (Table -3).The mucus was Woodman and Hammond [8] reported that the plentiful, fairly fluid and adhesive, containing no functional state of ovaries and their hormones had a cellular debris in both pregnant and non pregnant direct relationship with the consistency and elasticity animal at estrus.During the luteal phase the mucus of cervical mucus.became minimal, thickened, cohesive and contained When the animal conceived, its elasticity little cellular debris.These observations are in decreases sharply and it becomes thick and plastic, agreement with those of Woodman and Hammond [8], rather than elastic whereas in case of non pregnant Sharma and Tripathi [7], Sharma et al., [9].
animals the spinnbarkeit value decrease gradually [17].A defined trend in transparency and consistency Transparency and consistency did not show any change in pregnant and non pregnant cervico-vaginal remarkable difference in their pattern between mucus was not found in the present study.
pregnant and non pregnant animals during any phase of Mucus during estrus showed a particular type of estrous cycle.However, rheological properties elasticity which decreased as the estrous cycle (elasticity) showed a marked difference.cm), whereas in non pregnant group, the reduction was

Table - 1
. Details of samples studied.

Table - 2
. Colour of cervico-vaginal mucus in pregnant and non pregnant animals.Day 0 = day of estrus, Day 3-21= days of estrous cycle But on other cm).Whereas, in non pregnant group, the reduction days, differences were non significant.Hence it was was less and reached to 8.4±0.43 cm.The difference in concluded that though color and consistency were not spinnbarkeit values between pregnant and non helpful but a further and more detailed study of mucus th was observed on 9 day in pregnant animal (3.9±0.45 than in non pregnant (8.4±0.43)(Table-4).th pregnant group was found significant on 9 day of elasticity may pave a way for development of early estrous cycle.The mean spinnbarkeit value of cervical pregnancy diagnosis tool in buffaloes.

Table - 4
. Elasticity of cervico-vaginal mucus in pregnant and non pregnant animal.Unit-length of rope (in centimeter) formed on dropping some mucus from catheter to glass slide .A., Marsh, W. E., Calvin, J. A., Etherington, W.As per our results, transparency and consistency G., Momont, H. W. and Kinse, M. L. (1994) Pregnancy attrition associated with pregnancy testing by rectal did not show any remarkable difference between palpation.J Dairy Sci, 77: 3382-3387.pregnant and non pregnant animals during any phase of 4. Thurmond, M. C. and Picanso, J. P. (1993) Fetal loss estrous cycle as far as physical properties are concerned.associated with palpation per rectum to diagnose pregnancy However, rheological property showed a marked in cows.J Am Vet Med Assoc, 203: 432-435.