4.6. Chemical composition of feeds
The chemical composition of wheat straw and green fodder offered to experimental cows was within the normal range as reported by previous workers (Negi et al. 2015; Kumar et al. 2019; Wadhwa and Bakshi, 2023; Yatto et al. 2023). The DM, OM, CP, EE, TA, NDF and ADF of the concentrate mixture was also observed to be consistent with the results obtained by earlier workers (Choudhary et al. 2022; Patir et al. 2023; Singh et al. 2023). The composition of herbal powder and pellets was similar, while herbal extract had lower EE, TA, NDF and ADF values.
4.7. Hb and haematocrit
The present investigation demonstrates a noteworthy increase (P < 0.05) in haematocrit levels in the HEX group following administration of herbal formulation, while levels of Hb remained consistent across all treatment cohorts. These findings align with the established normal range for cattle, as noted by Kaneko et al. (2008). This elevation in haematocrit may be attributed to the potential enhancement of blood circulation facilitated by the herbal mixtures, ensuring efficient transportation of red blood cells throughout the body. The observed rise in haematocrit values could also be associated with the presence of flavonoids and quercetin, known for their hematopoietic properties (Raja et al. 2011). Furthermore, it's noteworthy that the herbal formulation contains volatile compounds (Mendoza et al. 2019; Sánchez et al. 2021). Our findings corroborate those reported by Nanda et al. (2013), who documented a significant increase (P < 0.05) in haematocrit concentration in goats following supplementation with a polyherbal formulation. Similarly, Barkakati and Kalita (2020) observed a significant elevation in Hb levels with polyherbal formulation supplementation compared to the CON group in cattle. Choudhury and Sinha (2015) observed a significant increase (P < 0.05) in haematocrit concentration in rats with the addition of M. koenigii leaves extract at doses of 250–500 mg/kg BW compared to the CON group. Lee-Rangel et al. (2022) also reported a significant increase in haematocrit concentration in calves treated with a polyherbal phytogenic source.
4.8. Serum glucose and NEFA
The synthesis, breakdown, and transport of lipids in the body have all been demonstrated to be influenced by herbal formulations containing polyphenols and flavonoids, which may decrease the release of NEFA from adipose tissue and increase lipid clearance from the bloodstream, thus lowering circulating NEFA levels (Hashemzadeh- Cigari et al. 2014; Baghbadorani et al. 2022). Furthermore, the decreased NEFA concentration could be due to the presence of bioactive compounds in herbal formulations (M. koenigii and A. marmelos) that enhanced energy metabolism and nutrient absorption in cows (Lopreiato et al. 2020). The improved energy utilization could lead to a reduction in circulating NEFA levels, as the cow’s body is better able to use energy sources from the diet. Nevertheless, these herbs are known to have hepatoprotective properties, supporting the liver’s function and metabolism. A healthier liver might contribute to better lipid metabolism, reducing the release of NEFAs into the bloodstream (Adewuyi et al. 2005). The present results are in concurrence with Barjibhe et al. (2019), who reported that polyherbal supplementation to cows significantly reduced the NEFA concentration in serum. In addition, cows supplemented with herbal extract in the treatment group had a lower (P < 0.01) concentration of serum NEFA (Baghbadorani et al. 2022). Razo Ortiz et al. (2020) reported that supplementation of polyherbal mixture (Ashwagandha, tulsi, gooseberry and giloy) to lambs did not have any significant effect to glucose level. Lozano-Sánchez et al. (2021) also observed that there was no significant effect on serum glucose with supplementation of polyherbal supplement in finishing lambs. Hashemzadeh-Cigari et al. (2015) observed that glucose level was not affected, but serum NEFA reduced in cows supplemented with herbal mixture as compared to the CON group, despite their analogous energy balance. Although the precise mechanisms responsible for the lower NEFA levels in cows supplemented with the herbal mixture remain unclear, it is proposed that the herbal mixture may inhibit lipolysis, thereby improving lipid metabolism and enhancing metabolic status in periparturient cows by increasing insulin sensitivity.
4.9. Total protein, albumin, globulin and A: G ratio
Blood metabolites such as total protein, albumin, globulin, and A:G ratio were assessed as indicators of the general health and vitality of the animals. The results revealed that these blood metabolites did not show significant differences (P > 0.05) among the various dietary treatments. These observation align with previous research conducted by Birudu et al. (2020), who similarly found no significant changes in A/G ratio and albumin levels in rats treated with methanolic leaf extracts of A. marmelos compared to control groups. Adebajo et al. (2006) also observed no significant impact on serum total protein, albumin, and globulin levels in Swiss Albino rats when supplemented with methanolic extracts of M. koenigii at different dosage rates. However, contrary results were reported by Gandhi et al. (2012), who noted a significant reduction in total protein levels in rats treated with A. marmelos. Furthermore, Dhami et al. (2022) reported varying plasma protein concentrations among different animal categories upon bi-herbal therapy with M. koenigii and A. marmelos, with lowered levels observed in buffaloes and infertile heifers compared to cows. Rivero et al. (2012) observed an increase in plasma protein levels after adding an extract rich in phenols to lamb diets, consistent with the findings of Lee-Rangel et al. (2022), who observed increased serum protein concentration in calves treated with a polyherbal phytogenic source. Furthermore, Dorantes-Iturbide et al. (2022) found that lambs fed a dietary supplement with a polyherbal additive containing hydrolyzable tannins, flavonoids, and essential oils exhibited a significant increase in serum albumin concentration. The analogous values of serum proteins observed in our study may be attributed to the relatively short duration of the treatment (7 days) compared to previous experiments, which were conducted for varying durations.
4.10. Total cholesterol
The feeding of the herbal formulation to dairy cows did not show a significant effect (P > 0.05) among the treatments. In this study, supplementation with the herbal formulation had no effect on serum cholesterol levels, suggesting that the bioactive metabolites did not impact intestinal absorption or lipid synthesis in the cells of cows (Dorantes-Iturbide et al. 2022). However, Razo Ortiz et al. (2020), who also reported that supplementation of polyherbal mixture (Ashwagandha, tulsi, gooseberry and giloy) to lambs do not have any significant effect to serum cholesterol level. In contrast to our findings, Satheesh and Pari, (2008) reported that saponins extracted from A. marmelos reduce blood cholesterol levels by competing with cholesterol for binding sites or by interfering with cholesterol biosynthesis in the liver. Priyadarshinee et al. (2021) observed that M. koenigii and A. marmelos leaves supplementation in cows significantly reduced the total cholesterol in treatment groups. The comparable cholesterol concentration in present study may be attributed to the short duration (7 d) of herbal treatment.
4.11. Serum urea, creatinine and serum enzymes
A significant (P < 0.01) reduction was observed in serum urea concentration in the HEX and HPL groups, indicating a decrease in rumen protein breakdown. This suggests enhanced EAA absorption in HEX, HPL, and HP groups (Pathak et al. 2017; Wankhede et al. 2022). Moreover, the decrease in serum urea indicates minimal protein catabolism and normal kidney function (Roseler et al. 1993; Hosten, 1990; Shavit et al. 2012). The serum creatinine value was also significantly (P < 0.01) lower in treatment groups as compared to CON. This beneficial effect may be due to the presence of polyphenols, tannins, and flavonoids in the herbal formulations. These compounds exhibit antioxidant activity and anti-inflammatory effects, and provide cardiovascular health benefits, potentially aiding kidney health by reducing oxidative stress and inflammation in renal tissues. Previous studies have associated these compounds with potential protection against kidney damage (Arulselvan et al. 2006). The findings are consistent with the observations of Arulselvan et al. (2006), who found that blood urea and plasma creatinine decreased in treatment groups when experimental rats were treated with M. koenigii, which provides addition al evidence to prove the anti-diabetogenic property of M. koenigii leaf extract.
The primary serum markers for assessing liver changes include AST, ALT, total protein, and total albumin were unaffected by the treatment involving herbal formulation mixture, indicating no liver damage (Díaz Galván et al. 2021). Furthermore, its polyphenols might provide an antioxidant benefit (Biswas and Giri, 2015), along with other advantageous effects on liver function due to this nutrient (Mehedint and Zeisel, 2013).
4.12. Serum mineral profile
Macro and micro elements play a crucial role in maintaining immune function and overall health. The high magnesium content in curry leaves suggests that they could serve as an excellent source of magnesium, which is known to activate various enzyme systems (Olusanya et al. 2008). Ca and P are also critical, as they influence an animal's ability to utilize other micronutrients and can modulate reproductive hormone sensitivity through various enzyme systems (Dutta et al. 2001). This could explain why the supplementation of curry leaves might lead to higher serum Ca and P levels and, in turn, support ovarian activity in anestrous heifers. Additionally, both M. koenigii and A. marmelos leaves are rich in essential minerals such as C, P, iron, zinc, manganese, and copper (Shantala and Prakash, 2005; Igara et al. 2016; Vijayalakshmi and Venkatalakshmi, 2017; Kujur et al. 2022). Our results align with Orzuna-Orzuna et al. (2021), who found that serum Ca levels among treatment groups were within the normal range for ovine, indicating that herbal mixtures do not disrupt mineral balance or nutritional status in lambs. In a similar vein, Sathesh Kumar and Punniamurthy (2009) observed that daily supplementation with 100 gd− 1 of M. koenigii leaves for 30 days significantly improved serum inorganic i-P levels and induced estrus in anestrous heifers. Similarly, Razo Ortiz et al. (2020) found that serum calcium levels were not affected by polyphenol and flavonoid doses, but serum phosphorus concentrations rose with increased doses of these compounds. Dhami et al. (2019) reported that supplementing cows with a mixture of M. koenigii and A. marmelos led to significant increases in plasma Ca, i-P, and other micro-minerals such as Zn, iron, and Cu. It is obvious that the mineral composition of the tree foliage is superior to that of tropical grasses. Goodchild and McMeniman (1994) reported that inclusion of tree foliage in ruminants’ diet increased the supply of minerals to the animals and rumen microbes and ultimately improved their performance. However, in contrary to our findings, Baitule et al. (2016) noted significant increases in serum Ca levels on day 9, the day of estrus, and on day 8 after estrus onset in treated groups, possibly due to supplementation with A. marmelos and M. koenigii. Likewise, Kumawat et al. (2014) found significantly higher serum Ca levels on days 4, 6, 8, and 10 after treating delayed pubertal heifers with dried leaf powder from A. marmelos and M. koenigii, compared to non-treated groups. This study also observed that the effect of these leaves on serum P levels was significant, with marked variations on days 2, 4, and 8 after treatment compared to non-treated heifers.
4.13. Reproduction
Supplementation of herbal formulation in different forms showed considerable improvement on confirmed pregnancy rate in crossbred cows (Patton et al. 2007; Ambrose, 2021). The beneficial effects of herbal formulations (A. marmelos and M. koenigii) supplementation could be due to the presence of phytoestrogens, minerals, and antioxidants. These components might stimulate gonadotropin hormones from the anterior pituitary, potentially contributing to the restoration of cyclicity in anestrus animals (Dutt et al., 2010; 2011). The antioxidants present may have enhanced folliculogenesis by preventing ROS-induced apoptosis of oocytes (Vijayalakshmi and Venkatalakshmi, 2017). The phytoconstituents might have stimulated steroidogenic activity in the hypothalamus, promoted follicular growth, and facilitated steroid production, leading to the initiation of cyclicity (Jondhale et al. 2009a; Nandini et al. 2010). Additionally, the presence of bioactive compounds in the herbal formulation may have contributed to follicular development, particularly during the dominance phase leading up to ovulation, resulting in the resumption of estrus. In the present study, though the conception rate was significantly higher in all the treatment groups relative to CON, however, HPL and HEX groups showed better conception rate and established pregnancy than HP (herbal powder) group, which could be probably due to the less wastage and complete consumption of herbal powder in pellets forms as compared to powder (Wanapat et al. 2013). The findings of the current study align with those of Patel and Gupta (2020), who reported the highest conception rate (71.42%) in delayed pubertal heifers supplemented with M. koenigii, A. marmelos, and a mineral mixture. Additionally, M. koenigii, whether used alone or in combination with a mineral supplement and a low dose of GnRH injection, was found to be equally effective in inducing fertility in anestrus buffaloes (Umashanker et al. 2006). Delayed pubertal heifers have shown induced estrus behavior after being supplemented with A. marmelos combined with M. koenigii (Kumar et al. 2016; Kumawat et al. 2016). Baitule et al. (2016) found 75% oestrus induction after supplementing A. marmelos and M. koenigii for 9 days in anestrous buffaloes. Satheshkumar and Punniamurthy (2009) also studied that supplemental effect of M. koenigii leaves for a period of 30 days and found induced oestrus in 60% of the anestrus heifers. Mehrotra (2002) have also documented that M. koenigii has been found to enhance ovarian function in rats and improve the therapeutic response in acyclic goats and heifers, particularly regarding oestrus and conception. Tiwari et al. (2021) reported that the oestrus response was 83.33% in postpartum buffaloes when supplemented with a mixture of A. marmelos and M. koenigii. However, Das et al. (2012) observed 92.90% oestrus induction in delayed pubertal buffalo heifers after feeding of A. marmelos and M. koenigii herbs for 9 days. Das et al. (2016) and Kumawat et al. (2016) reported that 90.9% and 92.3% of delayed pubertal heifers, respectively, began cycling after being fed a herbal mixture. Additionally, they observed a pregnancy rate of 54.1% in the group treated with the herbal mixture, compared to 16.7% in the control group. Therefore, it may be speculated that favourable effects of herbal formulations (mixture of M. koenigii and A. marmelos) in terms of estrus induction, ovulation, established pregnancy and shorter time period taken in induction of estrus are exhibited as compared to individual plant treatment which could be a reflection of synergistic actions of herbs active principles (Nandini et al. 2010; Hedge et al. 2002; Mehrotra et al. 2005). The active principles seem to operate either through mimicking gonadotrophins activity or stimulating the central mechanism for endogenous release of gonadotrophins along with possibility of local action (Dutt et al. 2018).
4.14. Physical characteristic of cervical mucus
It is believed that the herbal formulations (mixture of M. koenigii and A. marmelos leaves) are having hormonal regulatory properties that might have supported the oestrous cycle and overall reproductive health cows. Since, this herbal formulation having antioxidant and anti-inflammatory properties which might have helped in reducing inflammation in the reproductive tract, promoting better cervical mucus production and a favourable environment for sperm transport (Dhankhar et al. 2011; Gahlawat et al. 2014; Nigam and Nambiar, 2015; Rautela et al. 2018). Our findings align with those of Dhami et al. (2022), who observed that administering a bi-herbal treatment (M. koenigii and A. marmelos) @ 200 g daily for 5 days resulted in improved mucus quality at subsequent next oestrus in infertile animals. On contrary, Rahi et al. (2013) reported that on feeding of herbal extract containing polyphenols have reduced the pH which may be due to decline in bacterial load and inflammatory process in uterus after treatment.
The fertility is associated with an arborization pattern, as in our study we found that typical fern pattern was having higher conception rate in treatment groups thereby it was observed higher conception rate with typical fern pattern in all the treatment groups. The enhanced conception rate in crossbred cows with typical fern pattern indicates higher sperm penetration (Rangnekar et al. 2002; Layek et al. 2013). Alkaline pH of cervical mucus is favourable for higher sperm motility. The increase in pH in the treatment groups had higher fertility rate and also suitable for uterine environment with maximum sperm penetration in alkaline pH (Pattabiraman et al. 1967; Rangnekar et al. 2002). Similarly, the treatment groups exhibited a higher conception rate, correlated with an increased spinnbarkeit value. Additionally, the conception rate was influenced by the arborization pattern, as a typical arborization pattern is indicative of a proper hormonal balance, which supports higher conception rates (Alena et al. 2008).4.10. Relative mRNA expression of PGR genes
The progesterone gene’s expression was associated with the progesterone hormone, which was likewise shown to be significantly greater in the treatment groups. The fold expression of PGR gene was significantly higher (P < 0.01) in HPL, HEX and HP groups as compared to CON. Progesterone is a crucial ovarian steroid that regulates the estrous cycle, maternal recognition of pregnancy, and gestation in farm animals (Spencer et al., 2004). Beyond these roles, progesterone is also vital for various reproductive functions, including the behavioral expression of estrus, ovulation, maintenance of uterine quiescence, embryo survival and normal parturition.
Conclusions
In conclusion, the herbal formulation exhibited a positive influence on both metabolic parameters and reproductive performance in postpartum crossbred cows. The observed improvements in blood parameters, and early onset of estrus suggest promising prospects for integrating herbal supplements in dairy cattle management. These findings underscore the potential of herbal formulations as a supportive measure for enhancing the overall health and fertility of postpartum cows.