Effect of Estrus Induction on Blood Biochemical and Mineral Profiles and Fertility Rate in Retained Fetal Membranes Affected and Normally Calved Riverine Buffaloes

The buffalo (Bubalus bubalis) is one of the highest milk producing animals. Primary factor which affects the productivity of a buffalo is poor reproductive efficiency may be because of delayed puberty, age at first calving, prolonged calving intervals, higher number of services per conception, longer calving to conception interval, seasonal anestrus, uterine infections and various types of obstetrical ailments (Sachan et al., 2019). Retention of fetal membranes (RFM) is one of the major postpartum reproductive disorders affecting profitability of buffalo production since it delays uterine involution and resumption of ovarian activity leading to postpartum anestrus (Selvaraju et al., 2005). The incidence of postpartum anestrus in Indian buffaloes varies from 19 to 74% (Tomar et al., 2002). Various research workers (Ravikumar, 2003; Jagadeesan et al., 2006) have satisfactorily induced estrus and ovulation in postpartum anestrus cows using Controlled Internal Drug Release (CIDR) device. Velladurai et al., (2014) treated the crossbred normally calved (NC) and RFM affected cows with ovsynch protocol and concluded that ovsynch programme influenced the blood biochemical constituents and improved the conception. However, the effect of RFM and estrus induction with CIDR treatment in buffaloes which were affected and treated for RFM on the blood biochemical constituents and mineral profiles and their correlation with fertility have not been reported in detail. Hence, the present investigation was undertaken to assess the influence of estrus induction with CIDR plus prostaglandin F_2α (PGF_2α) protocol on blood biochemical and mineral profiles and to correlate these parameters with fertility rate in NC and RFM affected riverine buffaloes.

An experiment was conducted in graded Murrah buffaloes which were selected from Large Animal Gynaecology Unit, Veterinary College and Research Institute, Namakkal hospital and Veterinary Dispensaries which are located in and around Namakkal district from 2017-2019. Buffaloes (n=64) at 45-60 days postpartum including 32 buffaloes treated previously during immediate postpartum period for RFM and 32 NC were selected. They were equally divided into 4 experimental groups as group I and II (RFM groups) and group III and IV (NC groups), respectively. Buffaloes of groups I and III were dewormed orally with Albendazole suspension (Vetcare Private Limited, India; 10 mg/kg b.wt.) at 45-60 days postpartum. Further, they were treated orally with TANUVAS mineral mixture (30-50 g/day/animal) for 15 days in the concentrate feed from 45-60 days postpartum. At 60-75 days postpartum, buffaloes of all the 4 groups were inserted with CIDR containing 1.38 g progesterone (CIDR, Eazi-Breed, Pfizer Animal Health, India) intravaginally and left in situ for 9 days. All buffaloes received an i.m. injection of 500 µg PGF_2α (Cloprostenol sodium, Pragma, Intas Pharmaceuticals Limited, India) at 24 hours prior to CIDR withdrawal. All the buffaloes were artificially inseminated twice at 48 and 72 hrs of CIDR removal, with good quality frozen thawed semen. Further, those buffaloes which failed to conceive and returned to estrus following breeding at induced estrus, were artificially inseminated twice at an interval of 24 hrs during subsequent estrus. Pregnancy was confirmed by per rectal examination at 60 days post-insemination. Pregnancy rate was calculated in percentage for induced estrus (first cycle), second cycle and overall of two cycles in all four groups. Blood was collected at the time of (i) selection of animals (ii) CIDR insertion (iii) PGF_2α injection (iv) first/fix timed AI and (V) at 10 days after first AI from all the animals. The serum glucose was estimated immediately and the sera samples were stored at -20°C until analysis of other blood biochemical parameters, viz., total protein, triglycerides and total cholesterol and minerals, viz., calcium and phosphorus.
       
The blood biochemical constituents were analyzed in UV-VIS double beam spectrophotometer (Systronics, Model 2202, India). Blood glucose level (mg/dl) was determined from the serum samples by the GOD/POD method (Glucose Oxsidase/Peroxidase). Total protein (gm/dl) was determined from the serum samples by the Biuret method. The triglycerides (mg/dl) level was determined by GPO (glycerol -3-phosphate oxidase) method. Serum total cholesterol (mg/dl) was determined by the method of Allain et al., (1974). Serum calcium (mg/dl) level was determined by Arsenazo-III method. Serum phosphorus (mg/dl) level was estimated by the UV Molybdate-End Point Assay. The completely randomized design (CRD) was followed for the experiment (Snedecor and Cochran, 1994) and the data collected were analysed using SPSS® 20.0 software package. Post hoc analysis was done by Tukey’s honestly significance difference. The Fisher’s exact test was used to analyze the data related to the conception rate.

Conception rate
 
In the current investigation (Table 1), estrus induction with CIDR plus PGF2α resulted in 81.25 and 50.00% overall conception rates in NC and RFM affected buffaloes, respectively. It indicated that CIDR plus PGF_2α was effective in augmenting fertility in postpartum buffaloes. In this study, the overall conception rate of first and second service ranged from 37.50 to 87.50%. Similar conception rates with CIDR plus PGF_2α were obtained by Zaabel et al., (2009) in buffaloes and Cevik et al., (2010) in cows. But andurkar and Kadu (1995) recorded 100% conception rate in CIDR plus PGF_2α treated buffaloes. However, Murugavel et al., (2009) reported only 27.30% conception rate in buffaloes following CIDR plus ovsynch protocol. In this experiment, the groups I and III buffaloes which were treated with combination of deworming, mineral mixture and CIDR protocol resulted in 62.50 and 87.50% overall conception rates, respectively, whereas groups II and IV, with CIDR protocol alone, had 37.50 and 75.00% overall conception rates. This result indicated that either in NC buffaloes or in RFM affected buffaloes, alleviation of worm infestation and supplementation of mineral mixture for 15 days could further enhanced the fertility rate in buffaloes as described by Thavani et al., (2012).
 

       
In RFM affected groups (groups I and II) the overall first and second service conception rate obtained was 50.00% which was lower than the overall first and second service conception rate (81.25%) obtained in NC buffaloes. It was evident that RFM might have caused uterine damage and ovarian acyclicity which in turn might have reduced the conception rates following estrus induction as explained by Kimura et al., (2006).
       
The overall conception rate of 75.00% in group IV proved the efficacy of CIDR plus PGF_2α in improving fertility in buffaloes. The increased conception rate might be due to the fixed time breeding of buffaloes (Zaabel et al., 2009) and altered secretion of oestrogen and progesterone (Singh et al., 2010) following CIDR withdrawal. Fisher’s exact test revealed that only group II had statistically significant difference with group III. No significant difference was observed among other groups in conception rate.
 
Serum glucose
 
Blood glucose level exhibits a positive correlation with ovarian cyclicity in buffaloes (Singh et al., 2010). In the present investigation (Table 2), the blood glucose level was lower in RFM affected buffaloes than NC buffaloes. Similarly, low blood glucose level was reported in RFM treated buffaloes when compared to NC buffaloes (El- Malky et al., 2010; Thavani et al., 2012). In this investigation, from the time of selection of buffaloes to 10 days post AI, the mean blood glucose level was found to be low in RFM affected buffaloes when compared to NC buffaloes. This fact was reflected in conception rates between RFM affected and NC buffaloes. Velladurai et al., (2014) stated that blood glucose is a metabolic signal providing information for the central control of GnRH release and further reported that expression of estrus at first postpartum ovulation was more likely in cows which maintained a higher blood glucose level. The increasing concentration of blood glucose from animal selection to 10 days post-AI in the current experiment might be due to the increased energy metabolism to make the availability of glucose in the genital organs especially in the uterus as explained by Pandey et al., (2007).
 

 
Serum total protein
 
In this experiment, from the time of selection to 10 days post-AI, the mean serum total protein ranged from 8.09±0.23 to 8.56±0.21 mg/dl (Table 2).  Similar finding was reported in buffaloes by others (Jagadeesan et al., 2005; Jagadeesan et al., 2006; Kumar et al., 2010). In this study, at the time of selection of animals, the total protein levels did not show any significant difference between RFM affected and NC buffaloes. Higher concentration of serum total protein in RFM affected buffaloes than in NC buffaloes were reported by other scientists (Pandey et al., 2007; El-Malky et al., 2010). The increasing concentration of serum total protein from selection to 10 days post-AI might be due to the increased metabolism for the preparation of histotrophic nutrition in the uterus for the embryonic development (Cevik et al., 2010).
 
Triglycerides
 
The mean serum triglycerides in this study ranged from 83.83±1.51 to 131.89±0.59 mg/dl (Table 3). Similar values were reported by Velladurai et al., (2014) in cows.  However, lower level of serum triglycerides was reported by Singh et al., (2010). In this investigation, significantly low levels of mean serum triglycerides were observed in RFM affected groups than in NC groups. The reduction in mean serum triglycerides levels in this experiment might be due to disturbed lipid metabolism and increased tissue lipolytic enzymes in RFM affected buffaloes as demonstrated by Ravikumar, (2003). Induction of estrus in all the buffaloes increased the mean serum triglycerides from selection to 10 days post-AI. This might be due to the altered lipid metabolism towards the conception in buffaloes (Michal et al., 2006).
 

 
Serum total cholesterol
 
The mean serum total cholesterol ranged from 152.98±6.63 to 173.97±3.21 mg/dl (Table 3). Similar value was reported by Velladurai et al., (2014) in cows. Whereas Jagadeesan et al., (2006) found significantly low blood cholesterol concentration (119.89±3.32 mg/dl) in buffaloes before treating with CIDR. In this experiment, the mean serum total cholesterol level was lower in RFM affected buffaloes than NC buffaloes at the time of selection. Similar finding was reported by El-malky et al., (2010). The lowered cholesterol in RFM affected buffaloes might be due to the increase in breakdown of cholesterol (Velladurai et al., 2014). In this study, there was an increase in serum total cholesterol level in buffaloes from the time of selection to 10 days post-AI and it might be due to persistent utilization of serum cholesterol for progesterone synthesis (Michal et al., 2006).
 
Calcium
 
The mean serum calcium level recorded in present study ranged from 6.42±0.56 to 10.97±0.17 mg/dl in NC and RFM affected buffaloes (Table 4). Similar values were reported in NC (Jagadeesan et al., 2005) and RFM affected buffaloes (Pandey et al., 2007). In our study, the mean serum calcium level during all the stages of blood collection was lower in RFM affected groups than in NC buffaloes. This observation corroborated with the findings of Pandey et al., (2007) in buffaloes. It suggested that the lower calcium concentrations might be responsible for the occurrence of RFM in buffaloes and further reduction in fertility. The disturbances in the calcium metabolism and its utilization by the tissue result in atony of genital organs especially the uterus (Pandey et al., 2007). In the current experiment, the mean serum level of calcium increased in all the groups from the time of animal selection to 10 days post-AI. It indicated that estrus induction programme influenced the mineral metabolism of the buffaloes and increased the calcium availability to reproductive organs.
 

 
Phosphorus
 
The mean serum phosphorus level observed in this study ranged from 4.00±0.14 to 5.37±0.15 mg/dl in NC and RFM affected buffaloes (Table 4). In this study, higher levels of phosphorus in RFM affected buffaloes than in NC buffaloes were observed. Velladurai et al., (2014) stated that even marginal deficiency of phosphorus was sufficient to cause disturbances in pituitary-ovarian axis without manifesting the syndrome. The involvement of phosphorus in phospholipids and cAMP synthesis might be a key factor to its effect of reproduction (Ravikumar, 2003). In the current study alteration in calcium: phosphorus ratio was observed between NC (2:1) and RFM affected buffaloes (1:1). It clearly indicated the disturbance in calcium and phosphorus metabolism in RFM affected buffaloes as described by Pandey et al., (2007). The increased level of phosphorus from the time of selection to 10 days post-AI in all the experimental groups of the study showed that estrus induction programme with or without deworming and mineral mixture supplementation influenced the mineral metabolism in NC and RFM affected buffaloes and altered the calcium: phosphorus ratio at the time of AI as 2:1. It could be the reason for the achievement of higher fertility rate following estrus induction in reverine buffaloes of this study.

It is concluded that in RFM affected buffaloes estrus induction with CIDR plus PGF_2α changed the blood biochemical milieu, corrected the calcium and phosphorus ratio and improved the conception rate.

The authors are thankful to the authorities of Tamil Nadu Veterinary and Animal Sciences University, Chennai for the facilities provided to carry out this work.