Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 9 (september 2022) : 1077-1083

Effect of Janova, Sepia and Ovsynch Protocol on Blood Biochemical Profile and Fertility in Postpartum Anoestrus Cows

Jitendra Kumar1, Sushant Srivastava2, Rajesh Kumar2,*, Govind Mohan3, Vikas Chaudhiry2
1Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Nana Ji Deshmukhn Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.
2Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj-224 229, Ayodhya, Uttar Pradesh, India.
3Division of Animal Genetics and Breeding, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India.
Cite article:- Kumar Jitendra, Srivastava Sushant, Kumar Rajesh, Mohan Govind, Chaudhiry Vikas (2022). Effect of Janova, Sepia and Ovsynch Protocol on Blood Biochemical Profile and Fertility in Postpartum Anoestrus Cows . Indian Journal of Animal Research. 56(9): 1077-1083. doi: 10.18805/IJAR.B-4225.
Background: Anoestrus is still one of the most prevalent reproductive disorders in dairy cows despite technological advances in animal husbandry. Evaluation of blood biochemical profile is of diagnostic value to determine disease or health status of animal. The current study was aimed to evaluate the effect of different therapeutic regimen on blood biochemical profile, oestrus induction response and conception rate of anoestrus cows.

Methods: Thirty two postpartum anoestrus cows randomly allocated to four groups as G0 (negative control), GI, GII, GIII, GIV and 8 normal cyclic as GV (positive control). Dewormer and mineral mixture administered to GI, GII, GIII, GIV while group GII, GIII and GIV additionally received janova, sepia and Ovsynch protocol respectively.

Result: Different therapeutic protocols have variable effects on blood biochemical parameters. Overall oestrus induction response in G0, GI, GII, GIII, GIV and GV is 0.00, 50, 62.5, 75, 87.5 and 100.00 per cent respectively with corresponding conception rate of 0.00, 75, 80, 66.66, 57.13 and 75.00 per cent. On the basis of findings it can be concluded that aforesaid therapeutic regimens have definite bearings on fertility and can be used to manage postpartum anoestrus in cows.
Anoestrus is still one of the most prevalent reproductive disorders in dairy cows despite technological advances in animal husbandry. The anoestrus causes huge economic loss due to prolonged calving interval, reduced calf crop and shorter productive life. Incidence of anoestrus has been reported from 5.40% to 65.00% (Butani et al., 2008; Ashoo et al., 2020). There is general consensus that in dairy cows the anoestrus period after calving should not exceed 60 days.
       
The main causes of true anoestrus are low plane of nutrition (Dhami et al., 2017; Kumar et al., 2020a), chronic or debilitating diseases, senility, seasonal changes (Kalmath and Swami, 2019), heavy milk yield and management errors. To resume cyclicity postpartum threshold LH concentration is necessary, prolonged period of ovarian inactivity and failure of ovulation is mainly due to subthresold plasma LH level. Negative energy balance, malnutrition, stressors, deficient endogenous opiod peptides, suckling and lowered insulin concentration are risk factors for suppressed LH pulse (Patil et al., 1992). Battery of therapeutic strategies has been tried to manage postpartum anoestrus in cows (Kumar et al., 2011; Dhami et al., 2015; Naikoo et al., 2016; Dhami et al., 2017; Dhami et al., 2019; Mangrole et al., 2019; Satapathy et al., 2019; Vala et al., 2019) however, homeopathic therapy gaining popularity by virtue of its cost effectiveness and devoid of untoward side effects. Evaluation of blood biochemical profile is of diagnostic value to determine disease or health status of animal. The study was planned to assess the effect of different therapeutic regimen on blood biochemical profile, oestrus induction response and conception rate of anoestrus cows.
Selection of experimental animals
 
The present study was conducted at Department of Veterinary Gynaecology and Obstetrics, C.V.Sc. and AH ANDUAT, Ayodhya, in year 2016, with plan to develop economic and effective therapeutic regimen for postpartum anoestrus cows. The postpartum anoestrus cases presented at Veterinary Hospital Khandasa block and villages in adjoining areas of university were selected for the study.

The postpartum anoestrus cows were selected on the basis of history, breeding records, trans-rectal palpation and ultrasonography of genitalia. The cows which have not exhibited apparent clinical sign of oestrum, normal genital tract with atonic uterus and smooth, small inactive ovaries for at least 90 days postpartum were selected as anoestrus cows. Trans-rectal palpation and ultrasonography of ovaries were performed twice at ten days apart to rule out presence of follicle or corpus luteum on the ovaries and to confirm the cases of anoestrus.
 
Experimental design
 
The selected anoestrus cows (n=32) were randomly allocated to 4 groups as G0, GI, GII, GIII and GIV; comprising of eight (n=8) anoestrus cows in each group and normal cyclic cows (n=8) was assigned as GV (Table 1). The group G0 (anoestrus cows) and GV (normal cyclic cows) was kept as positive and negative control, while cows in group GI, GII, GIII were administered with fenbendazole @7.5 mg per kg body weight orally only once to reduce worm load and chelated mineral mixture powder @ 50 gm orally once in a day for 20 days. The cows in group II, III and IV were also treated with janova, sepia and ovsynch protocol respectively.
 

Table 1: Treatment protocol.


       
The cows of all groups were followed for oestrus detection in morning and evening and if found in standing estrus inseminated with good quality frozen thawed semen. The pregnancy was confirmed by trans-rectal palpation 2 month post AI in non-returned cows.
 
Blood sampling
 
Approximately 10 ml of blood was collected aseptically in a clean sterile glass test tube from jugular vein puncture using 16G sterilized needles from each anoestrus cows and regular cycling cows. The sample was kept in clean sterile glass test tube and tube was kept in slanting position away from sun light and allowed to clot. The serum was separated and stored in sterilized vials at -20°C until biochemical analysis.
 
Determination of biochemical parameters
 
Reitman and Frankel (1957) method was use to estimate serum glucose level (with the help of Span Cogent Diagnostic Kit, Span Diagnostics Ltd.) this method has been standardized with Kinetic Method (Standard Karmen Unit assay). Wabenga et al., (1970) method was used to estimate serum cholesterol (Span Diagnostic Ltd., India). Modified Biuret method was used for total serum protein estimation (Span Diagnostic Ltd., India). Bromocresol green method (Span Diagnostic Ltd., India) was used for estimation of serum albumin (Doumas et al., 1971). Serum calcium and inorganic phosphorus were estimated by spectrophotometric methods (Span Congent Diagnostic Kits).
 
Oestrus induction response and conception rate
 
After treatment, all the animals were visually observed for expression of estrus at morning, afternoon and evening at least for 30 minutes and confirmed by per rectal examination. After detection of estrus, animals were inseminated two times at an interval of 12 hours with good quality frozen thawed semen. Pregnancy was confirmed for 60 days post insemination in non-return cases.
 
Statistical analysis
 
The data were analysed by using completely randomized design and Duncan’s NMRT or ‘t’ test (Snedecor and Cochran, 1989). Data represented as mean±SE and considered as significant at P<0.05.
Effect on blood biochemical parameters
 
Glucose (mg/dl)
 
It was observed that glucose level is significantly lower at pre-treatment state in all anoestrus cows (G0, GI, GII, GIII and GIV) as compared to normal cyclic cows (GV). The glucose level in group G0, GI, GII, GIII and GIV at post-treatment state shows increasing trend and differ significantly (P<0.05) (Table 2). Likewise, Agrawal et al., (2015) reported significantly lower mean serum glucose level in post partum anoestrus cow than those of cyclic cow (42.58±6.73 g/dl vs 73.7±10.69 g/dl).
 

Table 2: Effect of mineral mixture, Janova, Sepia and Ovsynch protocol on blood biochemical profile (mean±SE) in postpartum anoestrus cows.


       
Many researchers have suggested that lower serum glucose level is responsible for subfertility, anoestrus (Yadav et al., 1995), nutritional anoestrus (Kumar and Saxena, 2010) and reflects energy status and reproductive activity of animals. Glucose may be a metabolic signal providing information for control of GnRH secretion and appears to be centrally involved in the release of LH and this presumably reflects its role in modulating GnRH release. Available reports suggests that lower glucose level might be a cause of clinical syndrome of anestrus.
 
Cholesterol (mg/dl)
 
The pre-treatment cholesterol level is significantly (P<0.05) lower in all anoestrus cows (G0, GI, GII, GIII, GIV) than those of normal cyclic cows (GV). The cholesterol level in untreated anoestrus cows (G0) at periodic sampling did not differ significantly but significantly lower than normal cyclic cow (GV). The cholesterol level in pre-treatment sampling of group GI, GII, GIII, GIV was significantly (P<0.05) lower than post-treatment sampling, which suggest positive effect of given therapy on cholesterol level (Table 2). Similarly, Kumar et al., (2009) reported lower cholesterol concentration in anoestrus, suboestrus and repeat breeder crossbred cows as compared to normal cyclic cows. However, Virmani et al., (2011) reported declined in cholesterol levels as animal approached towards cyclicity. The utilization of cholesterol for optimum steroid hormone biosynthesis to maintain the cyclicity may be correlated with lower level of cholesterol in cyclic animal. Unlike present findings, Bhoraniya et al., (2012) and Virmani et al., (2011) reported higher cholesterol level in non-cyclic than those of cyclic one. Reduced level of steriodogenesis may be attributed to low levels of cholesterol.
     
Kumar et al., (2009) observed high level of cholesterol in normal cyclic than the repeat breeder cows without any significant difference between conceived and non-conceived groups. Moreover, Singh et al., (2006) observed significantly higher level of serum cholesterol at induced estrus.
       
However, Mahour et al., (2011) reported lower cholesterol level at induced oestrus as compared to anoestrus cows (118.90±10.2 mg/dl vs 125.01±9.65 mg/dl).
 
Total protein (gm/dl)
 
The mean value of total protein did not vary significantly in periodic sampling of group G0. Moreover, mean total protein values of periodic sampling in group GV also did not vary significantly, though the values are significantly higher (P<0.05) as compared to group G0. The mean total protein values at pre-treatment did not differ significantly between group GII, GIII and GIV but their corresponding values are lower at induced oestrus (Table 2). Thus, when animal moves towards cyclicity total protein level increased as compared to anoestrus cows.    
       
Like our findings Kumar et al., (2013) reported higher total protein level in cyclic cows as compared to anoestrus cows. Likewise, Virmani et al., (2011) reported lower protein level in anoestrus cows than those at induced oestrus (6.52±0.57 g/dl vs 8.61±0.64 g/dl). Similarly, Agrawal et al., (2015) reported higher serum protein concentration in cyclic than anoestrus animals (8.20±1.09 g/dl vs 6.58±1.04 g/dl). However, Mahour et al., (2011) reported higher serum total protein in anoestrus cow against induced estrus cows (7.29±0.31 gm/dl vs 6.36±0.48 gm/dl; P<0.05). Bhoraniya et al., (2012) observed lower total protein level in anoestrus cows than normal cyclic cow (5.89±0.23 vs and 6.20±0.20 gm/dl). Moreover, Pariza et al., (2013) reported lower mean total serum protein in anoestrus cows than control one (3.4±0.8 vs 5.2±0.8 gm/dl; P<0.05).
 
Albumin (g/dl)
 
The mean value of albumin in periodic sampling of normal cyclic cows (GV) did not differ significantly (P<0.05). Moreover, periodic value of albumin in also did not vary significantly in positive control (G0) but significantly (P<0.05) lower than negative control cows. The mean albumin values at induced oestrus (post-treatment sampling) in cows of GI, GII, GIII and GIV group were significantly higher than corresponding pre-treatment values (Table 2). Thus, when animal attains cyclicity the mean albumin level increased significantly in treatment group (GI, GII, GIII and GIV). However, Virmani et al., (2011) reported slightly elevated serum albumin in postpartum anoestrus cow than control group. Moreover, Mahour et al., (2011) reported significantly higher (P<0.05) serum albumin in anoestrus cows than induced estrus cows (3.25±0.12 gm/dl vs 2.61±0.18 gm/dl).
 
Calcium (mg/dl)
 
The mean calcium level in periodic sampling of negative control (GV) and positive control (G0) did not vary significantly (P<0.05) but significantly lower in positive control cows. The mean calcium values at induced oestrus (post-treatment sampling) in GI, GII, GIII and GIV were significantly (P<0.05) higher than corresponding pre-treatment values (Table 2). Thus, when animal attains cyclicity the mean calcium level improves significantly (P<0.05). Similarly, Agrawal et al., (2015) reported significantly higher (P<0.01) mean serum calcium concentration in normal cyclic cow than anoestrus cow. Unlike present findings, Virmani et al., (2011) and Bhoraniya et al., (2012) reported of lower serum calcium level in normal cyclic cow against anoestrus cow as 7.50±1.21 vs 9.27 mg/dl and 8.01±0.79 vs 9.67±0.23 respectively. Calcium sensitizes tubular genital tract for action of hormones (Kumar et al., 2020b) and  plays a key part in improving the number and size of ovarian pre ovulatory follicles as well as the ovulation rate.
 
Phosphorus (mg/dl)
 
The mean value of phosphorus in periodic sampling of G0 and GV did not vary significantly (P<0.05) within group but corresponding periodic values in positive control (G0) was significantly (P<0.05) lower than negative control (GV). The mean phosphorus values at induced oestrus (post-treatment sampling) in cows of GI, GII, GIII and GIV group were significantly (P<0.05) higher than corresponding pre-treatment values (Table 2). Thus, when animal attains cyclicity the mean phosphorus level increased significantly in treatment group i.e. GI, GII, GIII and GIV group. Similar trends reported by Agrawal et al., (2015). Moreover, the mean serum phosphorus value in anoestrus animals were similar to the observation of Agrawal et al., (2015) but lower than that recorded by Virmani et al., (2011) and Bhoraniya et al., (2012). Furthermore, Kumar et al., (2009) reported significantly higher the phosphorus level in conceived group against non-conceived one.
 
Oestrus induction response and conception
 
The oestrus induction response was highest in  GIV followed by GIII, GII and GI respectively while none of cow exhibit oestrus in Go (untreated anoestrus cows) during course of study which shows importance of different therapeutic regimen in induction of oestrus in anoestrus cows (Table 3). Return in cyclicity in GI might be attributed to supplementation of mineral mixture containing iron, manganese, selenium, zinc, cobalt and iodine and various essential amino acids which are known to normalize the reproduction function (Rabbie et al., 2010). Like our findings in GI, Sahatpure et al., (2016) reported similar oestrus induction response (50%) by using herbal heat inducer Prajana HS (3 capsule/day for 2 days orally) and trace mineral supplement tablet CoFeCu (1 tab/day for 20 days orally). The findings of oestrus induction responsein GI and GII is corroborated well with Chaudhiry et al., (2019) who reported similar oestrus induction rate in anoestrus buffalo heifers. However, Kumar et al., (2011) reported higher oestrus induction response (76.92%) with use of lugol’s iodine 0.25% solution 10 ml on os cervix and supplementation of injectible phosphorus, injectible vitamin A and oral cyclomin -7 bolus with mean oestrus induction interval of 26.70±4.52 days. Singh et al., (2006) reported 60% and 80% oestrus induction response by using injectible sodium acid phosphate (Inj. Urimin, 10 ml, im, alternate days for days) and Bolus Minerex (2 boli, daily for 6 days) respectively. Mathur et al., (2005) reported 80% and 66.66% oestrus induction rate with oestrus induction interval of 10.25 days and 21.00 days in Frieswal heifer and Sahiwal heifer respectively by using injectible vitamin A (6 lac IU per week for 2 week) and injectible phosphorus (injTonophosphan @ 2 g /heifer for 5 alternate days. Dutta et al., (2019) observed 83.33 oestrus induction response in anoestrus crossbred cows with nutritional supplementation of  bypass fat @ 150 g/day/animal for 20 days  (Fatomax, Intas Pharmaceuticals Limited, Ahmedabad, India), mineral mixture @ 30 g/day/animal for 30 days (Minfa Gold, Intas, Pharmaceuticals, Ahmedabad, India), injectible phosphorus @ 2 g, im, for three occasions at alternate days (Tonophosphan Vet, containing sodium salt of 4-dimethyl amino-2-methyl phenyl phosphinic acid, MSD-Animal Health, Pune, India) and injectible vitamin A @ 5 ml, im, alternate days for three occasions (Intavita H, Intas, Pharmaceuticals, Ahmedabad, India).

Table 3: Efficacy of Janova, Sepia and Ovsynch protocol on oestrus induction response in postpartum anoestrus cows.


       
The oestrus induction response in GII are in agreement with findings of Pugashetti et al., (2009). Janova (ingredient: Citrullus collocynthus, Piper longum, Piper nigrum, Zingiber officinale) is known to exert gonadotropin like action and synchronizes the release of FSH, LH and estradiol for inducing ovulatory estrus. Feeding of janova appears to stimulate the hyopthalmus- hypophyseal ovarian axis there by synchronizing the hormonal release and inducing estrus in early postpartum anoestrus period with comparatively higher conception rate (Singal, 1995). Unlike findings in GII, Sahatpure et al., (2016) reported lower oestrus induction response with use of herbal heat inducer estroform powder (7.5 g/day, 2 days)  and trace mineral tablets Mintrus (1 tab/day,  20 days) orally.
       
Unlike present findings in GIII, higher and lower oestrus induction response was reported by Kumar et al., (2004) and Rajkumar et al., (2006) respectively which is suggestive of promising effect of homeopathic remedy in the management of anoestrus in cows. In contrast to present findings in GIV, higher resumption of cyclicity was reported by De Jarnette et al., (2001). Variable response to ovsynch protocol was also reported by Naikoo et al., (2016). These differences resumption of cyclicity and conception rate might be due variation in breed, climate, method and frequency of estrus detection, presence of large follicle at the time of treatment, body condition and milk yield of the animal.
       
The conception rates at induced or first heat in GI, GII, GIII and GIV group were 25, 20, 16.66 and 14.28 per cent respectively with corresponding pooled conception rates of three cycles were 75, 80, 66.66 and 57.13 per cent (Table 4). In negative control cows (GV) the pooled conception rates were 75% and none of the cow in positive control resumed oestrus cycle and conceived. Conception rate in GI and GII is in consonance with Sahatpure et al., (2016) who reported comparable conception rate as 75% by using estroform powder and mintrus tablet and 66.67% with use of capsule Prajana HS and tablet CoFeCu. Unlike present findings in GIII with use of sepia (62.5% oestrus induction and 80% conception rate), Rajkumar et al., (2006) reported 100% oestrus induction response in anoestrus cows with  treatment to oestrus induction interval of 27.5±5.3 days and 54.5% overall conception rate with 1.83 services per conception with use of homeopathic complex (Calcarea phosphoric 30c, Aletris farinose 30c, Pulsatilla 30c, Aurum muriaticum natronatum 30c, Sepia  30c and Phosphorus 30c ) in equal proportion, 15 pills, twice in day for ten days. Williamson et al., (1995) also reported beneficial effect of Sepia 200c for prevention of post-partum anoestrus in dairy cows and lowering calving to conception interval. Chandel et al., (2009) also observed significantly higher oestrus induction response (71.42% vs 20%) and conception rate (78.95% vs 0.00) against control with treatment to oestrus induction interval of 8.36 days by using homeopathic combination “Heat-O-Gen” (Alteris Farlnosa 1M, Folliculinum 1M, Oophorinum 1M, Pitutry 1M) two tablets twice a day in anoestrus buffaloes. Bhoraniya et al., (2012) and Dhami et al., (2015) reported comparable conception rate with ovsynch protocol.
 

Table 4: Efficacy of Janova, Sepia and Ovsynch protocol on oestrus induction response and conception rate in postpartum anoestrus cows.

In conclusion, postpartum anoestrus in cows is a multifactorial problem and oestrus can be effectively induced with janova, sepia and ovsynch protocol.
The authors thanks to Dean, C.V.Sc. and AH for providing necessary facilities to conduct the research.
None.

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