Effect of mineral and vitamins supplementation on hemato-biochemical parameters and reproductive performances of Gir and Jersey cross-bred cattle

Introduction

Animal husbandry and livestock are highly potential sectors of Indian Agriculture or the backbone of the Indian economy, especially of rural economy. It provides a livelihood for millions of families in the country. Worldwide dairy herd fertility has been stated to have declined drastically during the last decades leading to significantly increased calving intervals. The principal component of an increased calving interval is the prolongation of the interval between parturition and insemination. Anoestrus denotes a state of complete sexual inactivity with no manifestation of estrus and is observed during certain physiologic conditions, such as before puberty, during pregnancy, and lactation, and in seasonal breeders. It is most often a sign of permanent or temporary depression of ovarian activity, caused by seasonal changes in physical environment, nutritional deficiency, lactation stress, and aging. Certain pathological conditions of the ovaries or the uterus also suppress estrus manifestation. Several deficiency diseases and metabolic disorders result in delayed maturity and subsequent infertility in dairy cattle. To maintain high milk output, cows should give birth at the shortest possible intervals, by taking account of post-partum uterine involution, anestrous, and the duration of pregnancy. These nutritional stresses can affect the reproductive system and may be manifested by delayed return to cyclicity and failure of the animal to become pregnant. Energy, protein, vitamins, and minerals play a significant role in the establishment of fertility and among vitamins A, D, and E, and like micro minerals such as copper (Cu), manganese (Mn), iron (Fe), and zinc (Zn) are being recognized as a significant contributor for reproduction in dairy animals. Normal levels of biochemical constituents and mineral levels are of utmost importance for maintaining the functional integrity of the reproductive system [1] . Changes in biochemical constituents and mineral levels are important indicators of physiological state [2] as well as the reproductive performances of the animals [3,4] . Blood has crucial importance for the maintenance of physiological equilibrium in the body 5 . The knowledge of hematological values is useful in diagnosing various pathological and metabolic disorders, which can adversely affect the productive and reproductive performances of cows [6] . Important reproductive disorders like anestrous and repeat breeding have adversely affected dairy industries 7 . Unlike other nutrients, living organisms cannot synthesize mineral elements and the availability of minerals also decreases with the maturity of fodder [8] therefore, judicious mineral supplementation is essential to the livestock for better production and reproduction. Livestock production is often badly influenced by mineral deficiencies and imbalances of micro-nutrients. Trace elements including Cu, Fe, Zn, Mn, and certain macro-elements like Ca, Mg, and P are very essential for normal livestock growth. Trace elements may function as co-factors, as activators of enzymes, or as stabilizers of secondary molecular structure [9] . Deficiency or excess of minerals like P, Cu, and Zn have been associated with subnormal fertility and anestrus conditions [10] . A high incidence of anestrous is associated with deficiencies in glucose, protein, albumin, and globulin [11] . The hydrolysis of cholesterol ester is essential for the synthesis of steroid-based sex hormones and its estimation may provide useful information on the functional status of the ovario-hypophyseal axis [12] . Taking into consideration of above facts, the present study was undertaken to evaluate the reproductive performances and also to elucidate certain hematological parameters and biochemical profiles before and after supplementation of vitamins and minerals during anestrous conditions of Gir heifers, Gir X Jersey cross-bred heifers, Gir cows, and Gir X Jersey cross-bred cows in order

Materials and Methods

The present study was conducted at Haringhata Farm Complex, Mohanpur, Nadia West Bengal. A total of 40 anestrous cattle of 10 each under different categories such as Gir heifers, Gir cows, Gir X Jersey cross-bred heifers, and Gir X Jersey cross-bred cows were used. The animals with no corpus luteum in the ovaries and inactive ovaries were included in the anoestrus group. All the studied animals were 3 years and above of age, medium in size having 175 to 250 kg body weight. The animals were kept under standard farm management practices. Additionally, they were fed daily with 3-4 kg of dry roughage and 20-25 kg of green roughage with clean water. The selected cattle were divided into two major groups as experimental as group I and control as group II. The experimental group comprised twenty-four (24) animals and was subdivided into four groups of 6 in each group. The control group was comprised of sixteen (16) animals with a sub-group of 4 no animals from each category. All the animals in the control and experimental group were confirmed as anoestrus following 3 successive gynecological examinations at an interval of 7 days revealing the presence of poorly-developed genitalia including small, soft, hard, smooth, and plane inactive ovaries with no corpus luteum. Hematological analysis: The blood samples (20 ml) were collected from all animals aseptically and 5 ml of blood was transferred in each sterilized vial containing 10 mg of EDTA per ml as an anticoagulant for estimation of hematological profiles. The remaining blood (15 ml) was taken for separation of serum. The animals of the experimental group were treated with 5 ml of Vitamin A-D-E intramuscularly at weekly intervals for a month and 10 ml of 20% Sodium Salt of 4-Dimethylamino-2-Methyl Phenyl Phosphinic Acid intramuscularly at every alternate day for the same period. Additionally, 30 gm of mineral mixture was given orally to each experimental animal for one month. At the end of the supplementation period, the blood samples were collected for haemato-biochemical studies. The hematological parameters like total erythrocyte count (TEC), hemoglobin concentration (Hb), packed cell volume (PCV), total leukocyte count (TLC), and differential leukocyte count (DLC) were studied by standard methods [13] . The contents of the above tube were mixed well and allowed to stand at room temperature for 5 minutes. The Optical Density (OD) of the test was measured and the Drabkinʼs solution (blank) at 540 mm was standardized on a digital colorimeter for analysis of hematological parameters. The packed cell volume, RBC, Hb%, and TLC were estimated by following standard procedure [14] . Blood smears were made on clean and grease-free glass slides at an angle of 450. The blood smears were stained using Leishmanʼs stain. The slides were kept in the microscope and examined first at low power and then under oil emersion for counting of neutrophils, eosinophils, lymphocytes, and monocytes by tally marking.

Biochemical analysis:

The serum biochemical parameters studied were glucose, total protein, cholesterol, calcium (Ca), magnesium (Mg), inorganic phosphorus (P), copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe). Analysis of blood samples was carried out with reagents and procedures supplied along with the kits (Trans Asia Bio-Medicals Ltd., Solan, HP, India). Blood serum total protein was analyzed as per the standard protocols -Biuret Method, endpoint. Blood serum total protein was calculated as total protein (gm%) = Absorbance of Test /Absorbance of standard X concentration of std. (gm%). The concentration of blood serum cholesterol was estimated with reagents and procedures supplied along with the kits and analyzed as per the standard prescribed methods such as Dynamic Extended Stability, CHOD -PAP Method (With LCF), and End Point. The concentration of blood serum glucose was estimated with reagents and procedures supplied along with the kits and analyzed as per the standard prescribed methods -GOD -POD Method, End Point. Serum calcium was estimated by Atomic Absorption Spectrophotometer (Perkin Elmer A Analyst 100). The serum sample was diluted to 1:40 with a 0.1% (W/V) lanthanum chloride solution adjusted to ensure that concentrations fell within the suitable absorbance range and were expressed as mg/dl or mg%. Serum phosphorus was estimated by diluting at the rate of 1:6 with de-ionized water by an auto-analyzer with the help of a phosphorus Arsenazo kit (Diasis Diagnostic Sistemler, Turkey). Serum magnesium was estimated by Atomic Absorption Spectrophotometer. The serum sample was diluted to 1:40 with a 0.1% (W/V) lanthanum chloride solution adjusted to ensure that concentrations fall within the suitable absorbance range. Serum samples were diluted to 1:6 with de-ionized water by using AAS (Perkin Elmer A Analyst 100). Descriptive statistics was used to estimate the blood parameters of dairy cows and Logistic Regression (LR) was used to analyze the effect of vitamin and mineral supplements on the onset of estrus in dairy cows using the SAS-9.2 software package.

Results and Discussion

Hematological analysis: The mean hemoglobin level in the control and experimental group of pre-medicated and postmedicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows showed no significant variation and the level of Hb% in anoestrus Kankrej cows were reported in the earlier studies 15 and similar findings were also opined by other workers 16 and reported that the hemoglobin concentration was within the normal range and it was non-significant after mineral supplementation (Table -1). The mean concentration of PCV (%) in the control group and experimental group of pre-medicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows did not vary significantly. The level of PCV (%) was estimated at 38.03±0.44 and 39.25±0.30 in the experimental group of pre-medicated and post-medicated Gir heifers respectively. Further studies revealed levels of PCV (%) were 30.61±0.70 and 32.8±0.90 in the experimental group of pre-medicated and post-medicated Gir X Jersey cross-bred heifers respectively and the statistical analysis showed no significant variation between the experimental group of pre and post-medicated Gir X Jersey cross-bred heifers (Table-1) and Similar findings were reported by the earlier workers in anoestrus Kankrej heifers [17] . The mean value of TEC (10 6 /µl) in the control and experimental group of pre-medicated anestrus Gir and Gir X Jersey cross-bred heifers and cows were almost similar and showed no significant variation between them. The mean value of TEC (10 6 /µl) in the experimental group of premedicated and post-medicated Gir heifers was 4.81±0.33 and 4.78±0.09 respectively (Table 1). The statistical analysis showed no significant variation between the experimental group of pre and post-medicated Gir heifers. The insignificant variation in cows and buffaloes was also reported by other researchers in similar studies 18,19 and besides that RBC concentration was also documented as non-significant in a similar study [16] . In the present study, the level of total leukocyte count showed that there was no significant variation in the level of TLC (cumm) between the control and experimental groups of pre-medicated anestrus Gir and Gir X Jersey cross-bred heifers and cows. The mean value of TLC (cumm) in the experimental group of pre and post-medicated Gir heifers were 7000±98.88 and 6895.83±105.92 respectively (Table 1). The statistical analysis showed no significant variation between the experimental group of pre and post-medicated anoestrus Gir heifers and the present findings were supported by the findings of earlier research workers [18,20] in anoestrus Jersey cross-bred cows. The mean value of neutrophil (%) in the control and experimental group of pre-medicated anestrus Gir and Gir X Jersey cross-bred heifers and cows were almost similar and showed no significant variation among them. The level of neutrophil (%) in the experimental group of pre-medicated and post-medicated Gir heifers was 31.66±0.80 and 31.87±0.60. The statistical analysis showed no significant variation among the experimental group of pre and postmedicated Gir heifers and the parallel observations in anoestrus buffaloes were also documented [19] . The mean concentration of eosinophil (%) in the control group and experimental group of pre-medicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows did not vary significantly. The levels of eosinophil (%) were estimated as 5±0.36% and 5.29±0.23% in the experimental group of premedicated and post-medicated Gir heifers, respectively and these findings showed no significant variation among the pre and post-medicated Gir heifers and the similar results were documented in anoestrus HF and Jersey cows 21 . The level of lymphocyte (%) among the control and experimental group of pre-medicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows showed no significant variations but in the experimental group of pre-medicated and post-medicated Gir heifers were found to be 58.0±0.68 and 58.25±0.50% respectively. The experimental group of pre-medicated Gir X Jersey cross-bred anoestrus heifers and post-medicated Gir X Jersey cross-bred anoestrus heifers did not vary significantly and these observations in buffaloes were also reported earlier [19] . The level of monocyte with mean value (%) in the control and experimental groups of pre-medicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows were almost similar and showed no significant variation (Table 1). The differences in monocytes in the experimental group of pre and post-medicated anoestrus Gir heifers were statistically non-significant and were found to be similar to those recorded in HF and Jersey cows [20,21] .

Serum biochemical analysis:

The level of serum glucose (mg%) in the control and experimental group of premedicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows were almost similar and showed no significant variation between them (Table -2) and nonsignificant variation among the pre and post medicated Gir heifers was also supported by the previous studies [22,23] in non-descript cows and buffaloes. The total serum protein (gm%) in the control group and experimental group of pre-medicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows did not vary significantly. The concentration of serum total protein (gm%) in the experimental group of pre and postmedicated Gir heifers was 6.61±0.17 and 6.90±0.15, respectively and it showed significant variation (p< 0.05) in the experimental group of anoestrus Gir heifers. The significantly higher level of serum total protein as observed in the experimental group might be due to medication of vitamins and minerals and therefore it showed significant variation (p<0.05) between the pre and post-medicated Gir cows (Table -2) and the findings were parallel with the results of earlier studies [24] . Further, the level of serum cholesterol (mg%) in the control and experimental group of pre-medicated anoestrus Gir and Gir X Jersey cross-bred heifers and cows were almost similar, and no significant variation was found among them. The concentration of serum cholesterol level slightly increased in experimental groups of post-medicated anoestrus Gir heifers and hence no variance was observed in pre and post-medicated anoestrus Gir heifers (Table-2) and earlier researchers have also documented the alike findings in Jersey cross-bred cows [25], [26] . The mean value of serum calcium (mg%) in the experimental group of pre and post-medicated anoestrus Gir heifers was 8.16±0.243 and 8.82±0.11, respectively but the study showed a significantly higher concentration of calcium (p<0.01) in post-medicated Gir heifers (Table -2) and therefore, minerals and vitamin-D supplementation played an important role in enhancing the serum Ca concentration in anoestrus animals [27,28] . Besides this, the present study showed a significantly higher level of serum phosphorus (p<0.01) in the experimental group of postmedicated anoestrus Gir heifers (Table -2) and this finding is parallel with the findings in the case of HF X Ongole Jersey cross-bred cows [29] and in Murrah buffaloes [30] . Further, the experimental group of pre-medicated and post-medicated Gir X Jersey cross-bred did not vary significantly [31] . The level of serum magnesium was 2.74±0.10 and 2.86±0.07 mg% in pre-medicated and post-medicated Gir cows, respectively and it showed a significantly higher level (P<0.01) of serum magnesium (Table -2). And was no significant variation among the pre-medicated and postmedicated Gir X Jersey cross-bred cows 32 . The present study showed that there was no significant variation in the level of serum copper (ppm) among the control and experimental groups of pre-medicated anoestrus Gir and Gir X cross-bred heifers and cows. However, the level of copper (ppm) was significantly higher (p<0.05) in postmedicated Gir heifers (Table -2), and highly significant serum copper in post-partum treated Jersey cross-bred cows was also observed [33] . The lower level of copper in the anoestrus group in the present study might be due to lower estrogen levels in anoestrus animals but this study also showed no significant variation in terms of copper availability between the pre-medicated and post-medicated Gir X Jersey cross-bred heifers and similar observation in anoestrus Kankrej heifers was also reported by earlier workers [25] . The mean value of serum zinc (ppm) in the control and experimental group of pre-medicated Gir and Gir X Jersey cross-bred heifers and cows was almost similar and showed no significant variation among them. Further, the level of zinc (ppm) did not vary significantly among the pre-medicated and post-medicated Gir heifers. The mean concentration of serum zinc (ppm) showed no significant variation between the pre-medicated and post-medicated Gir X Jersey cross-bred heifers (Table 2). The present findings are parallel to the findings of earlier studies in Jersey crossbred cows and buffaloes [34] . Significantly higher levels of serum zinc observed in post-medicated Gir cows might be due to the combined effect of vitamin and mineral supplementation on the fertility parameters and blood biochemical profiles. Statistical analysis showed that the mean of serum iron was significantly higher (p<0.05) in post-medicated Gir heifers. Further study revealed that the mean serum iron was significantly higher (p<0.05) in post-medicated Gir X Jersey cross-bred heifers (Table 2). Similar findings were reported in post-partum treated Jersey cross-bred cows [33] . This study showed serum manganese significantly higher (p< 0.05) in post-medicated Gir heifers [35] . The mean value of serum manganese (ppm) showed no significant variation among the pre-medicated and post-medicated Gir X Jersey crossbred heifers [34] .

Effect of mineral and vitamin supplementation on reproductive performances:

After supplementation of vitamins and minerals for 30 days to 24 experimental animals (Gir and Gir X Jersey cross-bred animals), 14 showed normal estrus symptoms. Four cattle (2 Gir heifers, 1 Gir X Jersey cross-bred heifer, and 1 Gir X Jersey crossbred cow) exhibited estrus within 7-14 days after medication. However, 8 animals (2 Gir heifers, 3 Gir cows, 1 Gir X Jersey cross-bred heifer, and 2 Gir X Jersey crossbred cows) exhibited estrus within 14 to 21 days and one Gir cow, one Gir X Jersey cross-bred cow also displayed the estrus within 21 to 28 days. The present study revealed that 60% of the heifers and cows were found to respond positively to the supplementation of vitamins and minerals, whereas 13% of the control groups of heifers and cows also showed estrus signs, and similar findings were also documented in the earlier studies [36,37] . Earlier studies showed that Copper (Cu), Manganese (Mn), and Zinc (Zn) through their superoxide dismutase enzyme activity play a significant role in ovarian steroidogenesis and luteal function. Phosphorous is found to be part of ATP and is synthesized from ADP and Pi. It is being utilized as a driving force for maintaining normal reproductive physiology [38] and previous studies also reported that Phosphorus (P) is associated with reproductive abnormalities in cattle and its deficiency induces anoestrus [12] .

Conclusion

This study was based upon certain hematological and biochemical parameters and also to elucidate the reproductive performances of anoestrus cattle breeds including Gir heifers, Gir X Jersey cross-bred heifers, and Gir X Jersey cross-bred cows on the administration of vitamins and minerals. The results in terms of hematologic and biochemical analysis showed relatively similar findings as suggested by the earlier workers. However, most of the hematological parameters revealed no significant level among experimental cattle groups. However biochemical analysis such as the total serum protein, cholesterol, calcium, magnesium, phosphorus, copper, iron, and manganese were found at significantly higher levels in postmedicated anoestrus cattle. This study established the variations in hematological and biochemical values manipulate reproductive physiology and hence, help diagnose the anoestrus conditions. Therefore, it is recommended that precise research should be conducted on detailed hematological and biochemical parameters to evaluate the reproductive performances of dairy cattle.

Acknowledgement

AcknowledgementThe authors are greatly thankful to the DEAN, Faculties of Veterinary and Animal Science, West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, and authors acknowledge Officer-In-Charge, Haringhata Farm Complex, Mohanpur, Nadia West Bengal for providing all the facilities for the conducting this research program.

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