Gyneco-clinical and microbiological characteristics of cervical mucus in repeat breeder crossbred cows following ceftiofur therapy

The present study was carried out to investigate gyneco-clinical and microbiological attributes of cervical mucus following treatment of repeat breeder crossbred cows (HF×K) with ceftiofur crystalline free acid @ 6.6 mg/kg body weight administer separately in two groups of cows through intramuscular (Group-I) and subcutaneous route (Group-II). Each group included 15 repeat breeder cows whose selection was random and based on history of conception failure, gyneco-clinical observation of genitalia and number of artificial insemination per conception. The findings envisaged that the oestrus duration, tonicity of uterus and number of artificial insemination were derailed from normalcy but post-therapeutic characteristics of all these traits improved in repeat breeder cows and resembled to normal cyclic cows. The bacterial load of cervical mucus of repeat breeder cows (3.1×108±0.65×108, Group-I and 3.6×108±0.72×108, Group-II) was significantly higher than normal cows (2.1×103, Group-III) which, however, decreased significantly at post-treatment oestrus in both groups of repeat breeders. E. coli, Staphylococcus, Klebsiella, Anthracoid and Streptococcus spp. were isolated from cervical mucus of pre-treatment groups of repeat breeders. Although, prevalence of microorganism varied but E. coli was predominant followed by Staphylococcus spp. However, all of them disappeared from post-treatment cervical mucus of repeater cows. It is concluded from the finding that ceftiofur therapy of repeat breeder cows was beneficial to restore normalcy of oestrus duration, doughy tonicity of uterus and reduction in total bacterial load in uterus.

Introduction

Any deviation in breeding rhythm results in progressive economic losses due to widening of the dry period, the calving interval as well lactation during the life time of the animals. Among the deviations of the reproductive origin, repeat breeding is one of the important and commonly encountered disorders in cross-bred cows, whose incidence has been reported from 5.5 to 33.33 percent in our country (Saxena, 2004) [27] . The affected animals have a regular oestrus cycle and appear normal on superficial clinical examination but fail to become pregnant following three or more breeding to artificial insemination /or natural service (Bartlett et al., 1986) [3] . The repeat breeding occurs due to defects in gametes, endocrine dysfunctions, infections, nutritional deficiency etc. which culminate to either the fertilization failure or early embryonic death as evidenced from earlier reports that these two factors account for 10-20 and 45 percent, respectively in overall conception failure in cattle (Diskin et al., 2008 and Inskeep et al., 2005) [9, 13] . During the oestrus apart from other changes, cervical mucus is produced by mucus secretary cells that line the cervical epithelium (Cortes, 2014) [6] . The consistency of mucus is visco-elastic and varies with the stages of oestrous cycle. It has profound effect on fertilizing capability of the spermatozoa. The cervical mucus at the time of ovulation provides a suitable environment for the maintenance of metabolic activity of spermatozoa and helps in its penetrability, migration and livability. Deviation in oestrual mucus properties from the normal may thus affect the spermatozoa adversely disrupting the fertilizing process and culminate into infertility.

Therefore, physical and biochemical properties of oestrual mucus need to be investigated in repeat breeders. The present investigation was, therefore, under taken on systemic administration of ceftiofur crystalline free acid to determine its efficacy for the treatment of repeat breeder cross-bred cow with the objective to study the pre-and posttherapeutic gyneco-clinical and microbiological characteristics of cervical mucus of repeat breeder cows.

Materials and Methods

Materials and Methods

Experimental Protocol 2.1.1 Selection of Experimental Animals

A total of 36 repeat breeders cross bred cows from Sherpura village of Banas Dairy Palanpur were selected randomly on the basis of gyneco-clinical history of regular oestrus and appeared normal on clinical examination but failed to conceive following three or more breeding to artificial insemination with semen of good quality. The selection was restricted further to those animals which did not have any disorder of ovulation but showed abnormal oestrual cervical mucus detected by white side test (Bhat et al., 2014) [32] .

Groups of Animals

The selected animals were divided in the three groups. The each treatment group was consisted of 15 repeat breeder cows while six normal cyclic cows were assigned to control group. Group I: The animals of this group received single dose of Ceftiofur Crystalline Free Acid (Excede, Zoetis Inc. 2605 East Kilgore Road, Kalamazoo, Michigan 49001, USA) @ 6.6 mg/kg body weight through intramuscular route. Group II: A total of 15 cows were categorized in this group to receive a single injection of similar preparation at the same dose rate by subcutaneous route. Group III: Six normal cyclic animals which conceived within three breeding were kept as control. No medication was given to these cows.

OEstrus Detection

The oestrus was detected by observing external signs of heat such as bellowing, frequent urination, presence of cervical mucus, edema of vulva and congestion of vulvar mucus membrane. Each cow identified oestrus was bred by artificial insemination using frozen-thawed semen of good quality.

Collection of cervical mucus

Oestrus cervical mucus was collected aseptically by rectovaginal technique from each oestrus cow before breeding. The external genitalia was cleaned with mild antiseptic solution and dried with cotton. The mucus samples were obtained by aspiration using a sterilized plastic sheath connected to a disposable syringe. The plastic sheath was followed perrectally to confirm its position in the cervical canal (Panangala et al., 1978) [22] . The first sample of cervical mucus from the repeat breeder cows was collected at the time of oestrus prior to commencement of treatment whereas subsequent sample was followed at the time of post therapeutic oestrus. The samples thus obtained were transferred to suitable containers for transportation in the wide mouth thermos containing ice crystals to the laboratory for physical and various biochemical constituents.

Parameters to be studied

The bovine cervical mucus is extremely inconvenient to handle with conventional laboratory apparatus (Gibbons and Sellwood, 1973) [11] . Therefore, samples were diluted with triple glass distilled water and thoroughly mixed by means of magnetic stirrer (Reddy, 1974) [25] .

Gyneco-Clinical Attributes

The oestrous cycle length, oestrus duration (Ahmed et al., 2016) [1] and number of AI/services were studied from the records and uterine health by per-rectal examination of genitalia whereas the post treatment oestrus duration was recorded by observing external sings of oestrus at six hrs interval.

Microbiology of Cervical Mucus 2.4.2.1 Bacterial Isolation

Cervical mucus samples obtained from treated and nontreated repeat breeder cows were transferred aseptically immediately to laboratory into the transport media subsequently, the streaking of the each mucus sample was done primarily for bacterial growth on Brain Heart Infusion Agar (BHI Agar, HiMedia) and left for incubation at 37 ºC temperature for 24-48 hrs. The identification of gram positive (+ve) and gram negative (+ve) bacteria was done by gram staining and biochemical test of 3% KOH. For further identification of bacteria colonies were transferred on MacConkey agar and Eosin Methylene Blue Agar (EMB Agar). Then used IMViC biochemical tests for confirmation of organisms (Gani et al., 2008) [10] .

IMViC test

The IMViC tests represented a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic or facultative anaerobic rod which produces gas from lactose within 48 hours. The term "IMViC" is an acronym for each of these tests. "I" is for indole test, "M" is for Methyl Red test, "V" is for Voges-Proskauer test, and "C" is for Citrate Test. The lower case "i" is merely for "in", as the Citrate test requires coliform samples to be placed "in Citrate".

Indole test

In this test, the organisms under consideration were grown in peptone water broth. It contained tryptophan, which under the action of enzyme tryptophanase converted to an Indole molecule, pyruvate and ammonium. The indole was then extracted from the broth by means of xylene. To test the broth for indole production, Kovac's reagent was added after incubation. A pink/red layer formation on top of the liquid was identification of positive results.

Methyl Red and Voges-Proskauer test

The MRVP broth was used in these tests for bacterial growth. After growth, the broth was separated into two different tubes, one for the Methyl Red (MR) test and another for the Voges-Proskauer (VP) test. The Methyl Red test detects production of acids formed during metabolism through mixed acid fermentation pathway using pyruvate as a substrate. The pH indicator Methyl Red was added to one tube and a red color appeared at pH's lower than 4.2, indicating a positive test. The solution remaining yellow at pH 6.2 or above indicated a negative test which means the butanediol fermentation pathway was used. The VP test uses alpha-naphthol and potassium hydroxide to test the presence of acetylmethylcarbinol (Acetoin), an intermediate of the 2, 3-butanediol fermentation pathway. After adding both reagents, the tube was shaken vigorously then allowed to stand for 5-10 minutes. The development of pinkish-red color indicated a positive test which means the 2, 3-butanediol fermentation pathway was used.

Citrate test

This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate as its sole carbon source. The agar contains citrate and ammonium ions (nitrogen source) and bromothymol blue as an indicator. The citrate agar is green before inoculation, and turns blue as a positive test indicator which reflected the utilization of citrate.

Result based on following table

Result based on following table

Bacterial Count

The bacterial count (cfu/ml) in the cervical mucus of repeat breeder cows was carried out the serial dilutions of sample by making as per standard technique of Diliello (1979) [8] . The 5 fold (10 5 ) dilutions of samples were made and plated in replicates of 3.

After dilution of the sample, 0.1 ml of diluted sample was taken and spreaded plate across the surface of a Brain Heart Infusion Agar (BHI Agar). The plates were allowed to dry before incubation at 37 ºC temperature for 24 hrs. Subsequently, colonies counted manually. In each of the plates which possessed no. of colonies between 30 and 300. The plates which did not have standard number of colonies on surface of BHI agar were discarded. The cfu/plate was read from a plate in the linear range, and then cfu/ml of the original calculated mathematically in consideration to dilution factor and no. of colonies grew on BHI agar by following formula: B = N×D B = number of bacteria N = number of colonies counted on a plate D = dilution factor

Statistical Analysis

The data were analyzed statistically to know the changes in cervical mucus of normal cycling and repeat breeding crossbred cows by using Post-hoc test of mean by Duncan's test (Snedecor and Cochran, 1994) [30] .

Results and Discussions 3.1 Gyneco-clinical attributes 3.1.1 Uterine Tonicity

The percentage of doughy uterus at pre-treatment oestrus was observed in 80.00 and 86.67 repeat breeder cows of Group-I and II, respectively whereas it was 16.67 per cent in normal cyclic cows. Following treatment of repeat breeders, the number of cows which had doughy uterus decreased and it was found in 13.33 and 6.67 per cent cows (Table 3.1).

Treatments Groups	—	No. of animals	Microbiology	—	—
			Bacterial spp. (%)		Bacterial load (cfu/ml) (mean± SE)
Group-I	Pre-treatment oestrus	15	E. coli	53.33	3.1×108 ± 0.65×108a (7.2×107-7.6×108)
			Staphylococcus spp.	33.33
			Klebsiella spp.	6.67
			Anthracoid spp.	6.67
	Post-treatment oestrus		0.00		0.00b
Group-II	Pre-treatment oestrus	15	E. coli	40.00	3.6×108±0.72×108a (6.7×107-8.3×108)
			Staphylococcus spp.	20.0.0
			Klebsiella spp.	13.33
			Anthracoid spp.	6.67
			Streptococcus spp.	20.00
	Post-treatment oestrus		0.00		0.00b
Group-III	Normal cyclic (control)	6	Streptococcus spp.	16.67	2.1×103 ±2.1×103b

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Microbiological investigation of cervical mucus in treatment and control groups

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On the other hand, a reverse trend was observed for tonicity of uterus at pre-and post-treatment oestrus. The percentage of tonic uterus in repeat breeders was 20.00 and 13.33 in Group-I and II, respectively which increased drastically to 86.67 and 93.33 at post treatment oestrus in both groups. The regaining of uterine tonicity in both treatment groups was almost similar to normal cyclic cows (86.33%).

The results pertained to uterine tone, length of oestrus cycle and duration of oestrus are depicted in Table 3.1. The repeat breeder cows had uterus of less tonicity in comparison to normal cyclic cows which, however, was improved at post treatment oestrus in repeat breeder cows of Group-I (86.67%) and II (93.33%). These findings are at par with normal cyclic cows of present study. The tonicity of uterus classified as pronounced, moderate/mild has been studied by various workers (Mehta, 1986) [18] . Duration to which the uterus remains tonic has also been reported in relation to status of ovary and uterus (Bhat and Bhattacharya, 2012) [4] . These findings indicated that duration of uterine tonicity decreased significantly when animal had normal ovulation but possesses infection in uterus. The uterus remains maximum tonic when oestrus is intense (Mehta, 1986) [18] . The post-treatment findings of present study are indicative of elimination of infection from uterus and thereby return of uterine musculature to normal texture which might be deviated in repeat breeders by bacterial invasion as reported earlier (Azawi et al., 2008) [2] .

Number of Artificial Insemination

Table 3.1 showed the average number of artificial insemination required for conception in different groups of cows. The repeat breeder cows of Group-I and II were found to have more number of artificial insemination (4.73±0.50 and 4.20±0.28). The difference between groups of repeat breeders was non-significant whereas at post treatment oestrus, the number of artificial insemination observed to decrease (1.78±0.22 and 1.50±0.18) which did not differ than that of artificial insemination in normal cyclic cows (1.00±0.00) but the difference between pre-and post-treatment average of artificial insemination was significant in Group-I (4.73±0.50 and 1.78±0.22) and Group-II (4.20±0.28 and 1.50±0.18).

Oestrus Cycle Length

The results pertained to length of oestrus cycle in repeat breeder cows have been presented in Table 3.1. The average length of cycle was found to be 21.53±0.95 and 21.27±0.68 days in repeat breeder cows of Group-I and II, respectively. Following administration of ceftiofur crystalline free acid through intramuscular (Group-I) and subcutaneous (Group-II) routes, the length of oestrus cycle reduced to 20.93±0.34 days in Group-II whereas it was observed more or less similar to duration of pre-treatment length in Group-II. The differences between groups of treatment were non-significant.

Duration of Oestrus

The average duration of oestrus in repeat breeder crossbred cows was longer in Group-I (44.40±2.86 hrs) and Group-II (48.00±3.31hrs), which reduced to 34.00±2.08 and 35.20±2.33 hrs following treatment in both groups (Table 3.1). The difference was significant (p<0.05) between preand post-treatment duration of oestrus in Group-I and II but succeeding oestrus was found non-significant in two groups of treatment (34.00±2.08 hrs and 35.20±2.33 hrs). In normal cyclic cows, the oestrus duration of 32.00±3.69 hrs was observed which did not reveal any significant difference than that of post treatment oestrus duration of both groups (34.00±2.08 and 35.20±2.33 hrs). Longer duration of oestrus in repeat breeder cows than normal cyclic animals is apparent from the results presented in Table 3.1. The difference was significant (p<0.05). The oestrus duration in repeat breeder cows of Group-I and II was shortened following treatment which also differed significantly but was not significant from the normal cyclic groups. The repeat breeder crossbred cows have been reported to show prolonged oestrus extended beyond 36-48 hrs (Singh, 1997 and Dadarwal et al., 2005) [28,7] which corroborate the present findings at pre-treatment oestrus whereas oestrus duration of 34.0±0.85 hrs is reported in normal crossbred cows (Qureshi et al., 2000) [23] which is agreeable to present observations too in normal cows. Normal duration of oestrus in various breeds of cattle had been reported as 2-30 hrs (Noakes, 2001) [21] and 12-28 hrs in crossbred cows (Roberts, 1986) [26] . The prolonged duration of oestrus is postulated to be caused by hormonal imbalance, though, the exact etiology and pathogenesis remains elusive. Dadarwal et al. (2005) [7] reported that supra-basal progesterone levels during oestrus might be an important factor in causation of prolonged oestrus which in turn results in repeat breeding syndrome. However, it remains indescribable in the present study as the efforts are not made to investigate hormonal profiles of repeat breeder animals.

Microbiological Investigation 3.2.1 Bacterial Load and Isolation

Total bacterial load and types of bacteria in cervical mucus of treatment and control groups are presented in Table 3.3. The total bacterial load of 3.1 × 10 8 ± 0.65×10 8 with range a range of 0.72-7.6×10 8 was found in cervical mucus of repeat breeder cows of Group-I while it was 3.6 ×10 8 ±0.72×10 8 with a range of 0.67-8.3×10 8 in repeaters of Group-II. The difference between two groups was non-significant but it was significantly higher (p<0.05) than that of normal cyclic cows (2.1×10 3 ±2.1×10 3 ). In comparison to higher bacterial load in pre-treatment cervical mucus, none of the post-treatment mucus samples showed bacterial growth in both treatment groups. The pre-treatment cultural isolation from cervical mucus of repeat breeder cows showed that E. coli, Staphylococcus spp., Klebsiella spp., Anthracoid spp. and Streptococcus spp. were among the types of organism whose percentage was 53.33, 33.33, 6.67 and 6.67, respectively in Group-I whereas corresponding figures were 40.00, 20.00, 13.33, 6.67 and 20.00 per cent in Group-II. In normal cyclic cows, only Streptococcus spp. was identified (16.67%) while none of the cervical mucus sample showed bacterial growth in mucus obtained during post-treatment oestrus in both groups. Bacterial isolation of the cervical mucus from repeat breeder crossbreds and normal cyclic cows yielded a wide range of isolates as depicted in Table 3.3. It is discernible from the results that E. coli was found in majority of mucus samples of repeat breeders followed by Staphylococcus spp. among the pathogenic groups of organism. The presence of Klebsiella spp., Anthracoid spp. and Streptococcus spp. was also identified in pre-treatment samples of cervical mucus in repeat breeder cows of Group-I and II whereas none of the mucus samples revealed presence of organism at posttreatment oestrus in these groups. The uterine microbiology of healthy cows and that developed uterine diseases has been studied by various workers (Joshi et al., 2013 and Zaman et al., 2015) [14,31] . The findings of these workers corroborate the isolation spectra of most organisms as discerned in present study. However, the present findings showed variation with respect to prevalence of different microorganisms than those reported earlier which might be attributable to various sites of genital organs to collect the samples (Azawi et al., 2008) [2] . Histo-pathological changes in uterus to extent of mild, moderate and severe depend upon type of bacterial. E. coli was reported abundantly to cause mild to moderate type changes in uterus whereas severe uterine changes by Arcanobacterium pyogenes (Azawi et al., 2008) [2] . Since E. coli was predominant over other bacterial isolates in repeat breeders prior to treatment, it might have alter uterine environment probably hostile to spermatozoa and thus resulting into conception failure and poor physical properties of cervical mucus. On the other hand reverse might be true at post-treatment oestrus in repeat breeder cow where none of mucus sample revealed the presence of organisms.

The bacterial load recorded in the present study was higher in repeater breeder cows than the normal cyclic animals (Table 3.3). It decreased significantly at post-treatment oestrus as discerned in repeat breeders of Group-I and II. Similar findings of decreased bacterial load at subsequent oestrus after treatment have also been reported (Kumar et al., 2013 and Rahi et al., 2013) [16,24] . However a wide variation was evident in decreased load of bacteria from different studies conducted earlier which is agreeable to the action of various antibiotics and their potency used to inhibit the bacterial organism in uterus. Further, such a decrease in bacterial load after treatment with ceftiofur might be attributable to migration of leucocytes in to uterine lumen which have a phagocytic action on micro-organisms.

Number of artificial insemination and conception rate

The overall conception rate was observed to 60.00 per cent in treatment Group-I whereas it was 73.33 per cent in Group-II. Out of 15 cows in Group-I, the first, second and third service per conception rate was 26.67, 20.00 and 13.33 per cent whereas corresponding figures for cows of Group-II were 40.00, 26.67 and 6.67 per cent, respectively. The maximum number of cows (4/15) and (6/15) were observed to conceived at first service in Group-I and II, respectively. Whereas the first service conception rate was 100 per cent in Group-III normal cyclic cows. The findings of overall conception rate in repeat breeder and normal cyclic cows (Table 3.5) revealed that all cyclic cows conceived to first service and resulted into 100 per cent conception. In repeat breeder cows also, the conception rate of 60.00 and 73.33 per cent was discerned in Group-I and II, respectively which is quite comparable to normal cyclic group. Besides, the first service conception was also higher in repeaters in Group-II than Group-I. The higher conception rate is documented to be associated with physical properties of cervical mucus as it has been reported to improve when cervical mucus is clear, stringy and possesses typical fern pattern (Lim et al., 2014 and Negash et al., 2014) [17,20] . Further, the present findings are agreeable to earlier reports of intrauterine antibiotic infusion of repeat breeder cows advocated following in vitro sensitivity test (Bhat et al., 2013 and Singh et al., 2015) [5,29] . However, a wide variation in conception was revealed from findings of these authors which might be attributable to difference in the pharmacokinetic characteristics of different antibiotics which have been reported to vary from one to another formulation: Gentamycin (Haddad et al., 1987) [12] , Enrofloxacin (Kaartinen et al., 1995) [15] , Oxytetracyclin (Morro, 1980) [19] . The higher conception rate in repeat breeder cows of Group-I is a fair indication of better bioavailability of drug used in the present study which in turn might have attained maximum concentration to eliminate/or kill microorganisms of uterus and thereby production of clear colored and thin consistency of cervical mucus to enable the spermatozoa for maintaining better viability and motility required for conception.

Figures and tables from the paper

Treatment groups	—	No. of animal	Gyneco-clinical Attributes	—	—	—	—
			Uterine tonicity (%)		No. of AI	Length of oestrus cycle (days)	Duration of oestrus (hrs)
			Doughy	Tonic
Group-I	Pre-treatment oestrus	15	80.00	20.00	4.73±0.50a (3.0-10.0)	21.53±0.95a (16.0-27.0)	44.40±2.86a (36.0-72.0)
	Post-treatment oestrus		13.33	86.67	1.78±0.22b (1-3)	21.67±0.29a (20.0-24.0)	34.00±2.08b (24.0-48.0)
Group-II	Pre-treatment oestrus	15	86.67	13.33	4.20±0.28a (3.0-10.0)	21.27±0.68b (16.0-25.0)	48.00±3.31a (24.0-72.0)
	Post-treatment oestrus		6.67	93.33	1.5±0.18b (1-3)	20.93±0.34ab (19.0-23.0)	35.20±2.33b (24.0-48.0)
Group-III	Normal cyclic (control)	6	16.67	86.33	1.0±0.00b		32.00±3.69b (24.0-48.0)

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Gyneco-clinical attributes of cervical mucus in treatment and control groups.

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Character	Sources of variation	d.f.	Sum of Squares	Mean Square	Cal F
Length of oestrus cycle	Between Groups	3	54.717	18.239	3.086*
	Within Groups	56	330.933	5.910
	Total	59	385.650
Duration of oestrus	Between Groups	4	2511.273	627.818	5.910*
	Within Groups	61	6480.000	106.230
	Total	65	8991.273

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Analysis of variance for gynecol-clinical attributes of cervical mucus in treatment and control groups

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Sources of variation	d.f.	Sum of squares	Mean square	Cal F
Between group	4	1.81×1018	4.5×1017	13.974*
Within group	61	1.97×1018	3.24×1016
Total	65	3.78×1018

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Analysis of variance for Microbiological (Bacterial load) of

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Groups	No. of animals	No. of service	No. of animals conceived	Conception (%)	Over all conception (%)
Group-I	15	First	4	26.67	60.00(9)
		Second	3	20.00
		Third	2	13.33
Group-I	15	First	6	40	73.33 (11)
		Second	4	26.67
		Third	1	6.7
Group-III	6	First	6	100	100 (6)

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Conception rate in following treatment in different groups of animals

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Groups	No. of animals	No. of service/conception	—	—
		First	Second	Third
Group-I	15	4 (26.67)	3 (20.00)	2 (13.33)
Group-II	15	6 (40.00)	4 (26.67)	1 (6.67)
Group-III	6	6 (100)	-	-

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Number of services per conception in treatment and control groups

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