Effect of Fat and Protein Along with Polyherbal Preparation on Reproductive Health of Periparturient Karan Fries Cows

Transition period in dairy cows is dominated by a chain of metabolic adaptations including dramatic changes in endocrine status to support lactation and avoid metabolic disorders (Pascottini et al., 2020). The practical purpose of the nutritional strategies during this timeframe is to support these metabolic changes as there is a sudden and marked increase in nutritional requirements for milk production when nutrient supply lags far behind. Some previous studies revealed that during pre-partum period in cows, high energy dense diet was associated with lower incidence of displaced abomasums and high protein content of this diet was associated with lower incidences of retained placenta and ketosis (Overton and Waldron, 2004). But, enhanced milk production capability paired with nutritional manipulation of diets has been accompanied by decline in fertility of lactating cows (Butler, 2003). Impairment of the immune status during the periparturient period in cattle increases the risk of uterine infections (LeBlanc, 2010) which are more prevalent during this phase of the reproductive cycle (Sheldon et al., 2002). Parturition and lactation induced stress increase susceptibility of dairy cows to infectious diseases and delayed ovulation (Butler, 2012, Khatti et al., 2017). Therefore, in spite of using antibiotics which comes along with the possibility of developing drug resistance, the need arise for the use of herbal feed supplements. The traditional Indian medicine system, Ayurveda, describes several herbs having active principles with antioxidant, antimicrobial and spasmolytic activities which are cheaper alternate and locally available to rural farmers for livestock health management. It is said that profitability of the cows during lactation is influenced by success of its transition period. The aim of this study was to develop a holistic approach or nutritional strategy by supplementation of fat and protein along with post-partum supplementation of polyherbal mixture to improve periparturient reproductive performance of transition crossbred cows.

Animals, diet and management
 
This study was conducted in the year 2017 at Livestock Research Centre of ICAR-National Dairy Research Institute (NDRI), Karnal, India, situated at an altitude of 250 m above mean sea level, latitude and longitude position being 29° 42” N and 79° 54” E, respectively. Twenty seven (dry, advance pregnant) Karan fries cows were selected from the LRC and distributed randomly into three groups of nine each based on their body weight and (MPPA) [T1 (control)= 420±20.1 kg, 3104±219]; (T2= 418±24.2 kg, 3035±210); (T3= 429±22.0 kg, 2865±210), respectively. The feeding experiment was carried out for 105 days i.e. 45 days before and 60 days after parturition. Each animal was provided an adequate floor space (length: 2.20 m width: 1.80m), tethered separately with rope and housed in a well-ventilated shed having facilities for individual feeding and watering facilities. Animals were offered experimental diets in the form of total mixed ration (TMR) after an adaptation period of 10 days. During pre-partum period, cows of group T1, T2, T3 were given TMRs having 13.1, 13.1, 16.1% CP and 9.82, 9.82, 10.8 MJ/kg ME, respectively. Whereas, during post-partum period, cows were offered TMRs containing 14.0, 16.8, 16.8% CP and 10.2, 11.0, 11.0 MJ/kg ME. Additional energy and protein in treatment groups was supplied by incorporation of full fat soya in the diet. Animals in T2 and T3 groups were supplemented with 215 g polyherbal mixture during post-partum period. Individual herbs were procured from local market, pulverized and mixed in a specific proportion. This polyherbal preparation (comprising Gloriosa superba, Rubiacordifolia, Tribulusterrestris, Artiolochindica, Nigella sativa, Solanum xanthocarpum, Stereospermum suaveolens, Cyperusscariosus, Tinosporacordifolia, Trigonella foenum-graecum, Arctium minus) was fed as top dressed to individual cows with concentrate and jaggery in the morning. Additionally anionic mixture pre-partum was also given to supplemented groups to avoid metabolic and reproductive disturbances associated with stress of high lactation which was being expected by effect of treatments. All animals were de-wormed for control of both internal and external parasites before the start of the experiment. The animals were fed twice a day and fresh clean water was given free of choice round the clock. This experimental study was done in compliance with appropriate laws and guidelines provided by the Institutional Animal Ethics Committee, New Delhi, MoEFCC, Government of India.
 
Immune parameters
 
Blood samples were collected in the morning before offering food, from the jugular vein in vacutainer tubes containing sodium heparin. Sample collection was done at -21,-14,-5, 0, 5, 14, 30, 45 day relative to parturition. Total immunoglobulin (Ig) in the plasma sample was estimated by Zinc turbidity method (Mc Ewan and Fisher, 1970). Total antioxidant activity was measured by ferric reducing antioxidant power (FRAP) assay of Benzie and Strain (1999).
 
Reproductive parameters
 
Animals were diagnosed with retained fetal membrane (RFM), if fetal membranes were retained for more than 12 hrs post-partum (Roberts, 1986). After parturition, animals were observed daily up to 15 days for lochial discharge, diagnosis of postpartum metritis and any abnormal discharge. All experimental animals were observed regularly for first sign of estrus. The interval between calving to successful conception was recorded as service period. Conception rate was defined as the proportion of cows gets conceived out of total inseminated cows.
 
Cows were examined rectally by same veterinarian using the ultrasonography technique (USG) for involution of uterus and animals suspected for incomplete involution were taken for diagnosis of endometritis. During USG procedure, the transversal diameters of the involuting uterine horn and cervix were measured using a real-time B-mode ultrasound scanner. Uterine involution was considered to be complete when no further reduction in the uterine diameter for three successive examinations was observed.  
Statistical analysis
 
The data were presented in mean ± standard error (SE) and effect of dietary treatments on various parameters was analyzed by one-way ANOVA using SAS (2003). The differences among means were considered statistically significant at 5% level of probability (P<0.05) and post-hoc comparison was performed using Tukey’s Studentized Range Test.

Immune status
 
Antioxidant status
 
In the current study, though cows showed individual variations in total antioxidant activity (µM/L) but no significant difference (P<0.05) was observed among different treatments throughout the experimental period in plasma total antioxidant activity except supplemented groups have numerically higher FRAP values (Table 1). On approaching parturition, the mean FRAP value decreased (Fig 1) due to more oxidative and hormonal stress in all treatment groups but relatively higher in control animals. Besides fulfilling the nutrient requirements during this transition phase, addition of more energy and protein might have supported in attaining better immune status by ameliorating the negative energy balance state in supplemented groups. Though antioxidant effect of this indigenous traditional knowledge based polyherbal mixture has not been reported earlier scientifically in dairy cows but antioxidant activity of individual herbal ingredients i.e., Nigella. sativa (Parhizkar et al., 2011), Trigonella foenum graecum (Bilal et al., 2003), Tribulus terrestris (Zheleva-Dimitrova et al., 2012; Hammoda et al., 2013; Sharma et al., 2013), Solanum xanocarpum (Ghassam et al., 2014), Articumminus (Duh 1998; Erdemoglu et al., 2009), Tinospora Cordifolia (Prince and Menon, 1999) in laboratory animals has been shown in various research studies. Similar to findings of present study, Mallick et al., (2011) reported no significant change in plasma antioxidant status of transition crossbred cows supplemented with Tinospora Cordifolia. Also, Chaudhari (2016) reported no significant difference in total antioxidant status of buffaloes during early lactating on supplementing high energy diets. However, Sharma et al., (2014) revealed improved antioxidant status of crossbred cows upon supplementation of polyherbal mixture (Withaniasomnifera, Ocimum sanctum, Tinospora cordifolia, Emblica officinalis, Nigella sativa, Tribulus terrestris and Asparagus racemosus) at the rate of 200-250 mg/kg body weight.
 

 

 
Plasma total Ig
 
On approaching parturition (-5d), plasma total Ig concentration (mg/ml) was found to be significantly higher in T3 group as compared to control (Fig 2). However, mean plasma total Ig concentration on the day of calving did not differ significantly (P>0.05) among various treatment groups but from 2^nd week onwards after calving, it varied significantly (P<0.05) from each other, T3 group being highest. Around calving, decrease in lymphocyte responsiveness has been observed due to poor antioxidant status of animal (Kehrli et al., 1989) which might contribute to  decreased plasma Ig concentration at the time of parturition. The active principles present in polyherbal preparations have immuno modulatory properties and enhance immunity as shown by many in vivo and in vitro studies (Maji et al., 2014; Tailor et al., 2010; Dikshit et al., 2000; Mallaiah et al., 2015). Further, the results of the study (Sharma et al., 2014) showed that polyherbal mixture supplementation reduced periparturient stress and improved immunity. Pre-partum supplementation of fat and protein might have improved the nutritional status of animal as dry matter intake decreases drastically during transition period and animal is not able to meet its increasing nutritional requirements, resulting in stress associated with negative energy balance. Likewise, Mustafa et al., (2016) reported lower concentration of immunoglobulin G in group fed lower metabolizable energy and protein level in transition buffalo diet.
 

 
Reproductive performance
 
The effects of various dietary treatments during transition period as well as on carry over period on reproductive parameters are shown in Table 2. Three out of nine cows in T1 group had shown incidence of retained placenta while T2 and T3 group cows had lower incidence of retained placenta although there was no case of calving difficulty in all the groups. Cows in supplemented group (T3) were recorded significantly lower (P<0.05) time required for expulsion of placenta as compared to control group animals. In the present study, cases of metritis and cervicitis are more prevalent in control groups i.e. three and two out of nine, respectively. The service period was reduced (P<0.05) in T3 and T2 group by 26 and 42 days than that of control, respectively. The conception rate was observed to be increased by 33.3 and 22.2% in T3 and T2 treatment groups in comparison with control group. No significant difference (P>0.05) was observed in birth weights of calves among various treatment groups.
 

 
Reproductive performance of dairy cows is mostly affected by retained placenta (Le Blanc, 2008) as it is associated with reduction in milk production, higher incidence of metritis and impaired fertility (Holt et al., 1989). It is established in many studies that immunosuppression which earlier required for maintaining pregnancy might be the cause of retained fetal membranes after calving thereby urging the need of immunomodulatory agents in order to maintain healthy reproductive environment of the body. Besides the immunomodulatory properties of polyherbal preparation, the antimicrobial and anti-inflammatory properties of herbs such as Nigella sativa (Yildiz and Balikci, 2016), Rubia cordifolia (Tailor et al., 2010), Tinospora cordifolia (Jeyachandran et al., 2003) provide more favorable environment and reduced pathogen invasion in reproductive tract of cows resulting in less number of cases of metritis and infections. This nutritional strategy post-partum could some extend replace the antibiotic therapy in producing animals. Higher retained placenta incidence in non-supplemented group might be the reason for higher incidence of metritis and cervicitis in these cows which in addition correlated to delayed commencement of cyclicity in these animals. The findings of first estrus after calving and service period indicate that cows in treatment group resumed cyclicity sooner after parturition. The synergistic effect of various herbs along with better nutritional inputs might have been resulted in better conception rate of cows in T2 and T3 groups. Also, various studies have revealed that upon supplementation of fat there was improvement in reproductive performance as higher conception rates (Ferguson et al., 1990), increased pregnancy rates (Schneider et al., 1988) and reduced open days (Sklan et al., 1991). Also, pre-partum higher energy intake in cows resulted in reduction in service period (Lopes et al., 2009). Supplementation of fat in cows has been thought to result in decreased PGF2α secretion from the uterus (Oldick et al., 1997) along with reduction in sensitivity of corpus luteum to PGF2α which might lead to increased conception rate in cows (Thatcher et al., 2006). However, in other study it was observed that supplemental fat has had little or no benefit on reproductive efficiency of the animal (Carroll et al., 1990).

The percent involution of pre gravid uterine horn during first two weeks of parturition was found to be significantly higher (P<0.05) in T2 and T3 groups as compared to control (Table 1, Fig 3). The mean values of percent involution of non-gravid uterus and cervix didn’t differ significantly (P>0.05) among treatments but data of supplemented groups supports the beneficial effect of dietary treatment on involution of uterus and cervix. The present findings revealed that involution of uterus and cervix was attained earlier in T2 and T3 group of cows as compared to the animals in T1 group. As per traditional knowledge and laboratory studies, various properties of individual herbs like Aristolochia indica as an emmenegogue, Cyperus scariosus and Gloriosa superba (Malpani et al., (2011) possessing spasmolytic activity, Tribulus terrestris having luteinizing effect (Dehghan et al., 2012), Solanum xanthocarpum having estrogenic activity (Aswar et al., 2014) might have played significant role in early expulsion of placenta and reduced cases of retained fetal membranes which would directly influence uterine involution period.
 

In summary dietary increase in nutrient concentration both pre and post-partum along with supplementation of polyherbal preparation post-partum improved reproductive and immunological performance in early lactating crossbred cows. Thus, this nutritional strategy during pre and post-partum period can be used for smooth transition and better health of crossbred cows.

We thank the Director, ICAR-NDRI, Karnal for providing necessary research facilities required during this study. We are also thankful to Dr. Nitin Raheja for his professional help in the experiment.