PLASMA HORMONES AND MILK PRODUCTION PERFORMANCES IN EARLY LACTATION BUFFALOES SUPPLEMENTED WITH A MIXTURE OF PRILLED FAT, SWEETENER AND TOXIN BINDER

: The effect of combined feed supplements (prilled fat, sweetener and toxin binder) was studied on 24 apparently healthy early lactating rural and urban maintained Murrah buffaloes. The feeding of combined feed supplement was carried out for a period of 90 days. DMI, BCS, body weight were recorded at fortnightly intervals and milk composition was analyzed at weekly intervals. Blood samples were analyzed for hormones, plasma metabolites and lipid profile. The supplementation increased (p<0.01) milk yield by 13.6 and 17.0% in urban and rural Murrah buffaloes with respective increases of 20.14 and14.98% in milk fat (p<0.01). BCS and DMI varied non-significantly (P>0.05) between the groups. Body weight increased in rural buffaloes in comparison to urban buffaloes. Plasma GH was higher (p<0.05) before supplementation and fluctuated non-significantly (P>0.05) during supplementation period. Mean leptin levels decreased (p<0.05) while plasma estradiol and IgG level increased during the supplementation period. Plasma progesterone and ghrelin level varied non-significantly before and during supplementation. Plasma IGF-1 and glucose levels was more and NEFA level was lower (p<0.05) during the experiment. Mean HDL, triglyceride and cholesterol concentration increased (P<0.05)


Introduction
Most of the animals in developing countries including India are being fed on agriculture by-products and low quality crop residues, which have inherent low nutritive value and digestibility. The requirement of energy is very high during early lactation but increased nutrient demand and limited feed intake due to physiological stage affects production potential of animal (Sirohi et al., 2010). Due to this reason animals are often forced to draw on body reserves to satisfy energy requirements (negative energy balance) leading to substantial loss in body weight and lower milk yield (Kim et al., 2003). Cereal grains and fats play an important role as source of energy in the ration of high yielding dairy animals for optimum productivity. However use of cereals for human consumption and monogastric animals, the alternate source of energy in the form of bypass fat in dairy ration has been reported (Saijpaul et al., 2010;Singh et al., 2015). Inclusion of unprotected fat in dairy ration up to 3% of dry matter (DM) intake, reduces digestibility of fibre (NRC,2001) and depresses rumen cellulolytic microbial activity (Ranjan et al., 2012). Supplementation of bypass fat increases energy intake and unsaturated fatty acid content of buffalo milk and provide more economic returns to dairy farmers (Parnerkar et al., 2010). The experiment on effect of prilled fat containing vegetable palm oil have shown significant impact on milk yield, fat percent and reproductive performance in cows and buffaloes of organized herd Yadav et al., 2015;Singh et al., 2015a). Since prilled fat feeding does not affect digestibility of feed and improves the reproductive performance, its effect on milk production and reproductive performance needs to be investigated in buffaloes maintained in tropical condition. In present investigation the effect of a supplementation mixture containing prilled fat, sweetener, toxin binder on milk production, composition, lipid profile and hormones was studied in rural and urban Murrah buffaloes. Sweetener was added to enhance the palatability of feed. Further, to determine its economic impact the cost benefit ratio of feeding was also determined.

Experimental design, diets and procedures
Lactating Murrah buffaloes (24) on day 38 postpartum in 2 nd -3 rd parity were selected from Gohargarh rural village (group I) and progressive urban dairy farm (group II). Buffaloes of both the group were supplemented with a mixture of prilled fat, sweetener and toxin binder, 100gm, 30gm and 20 gm/d respectively for aa period of 90 days. The experiment included two weeks observations prior to supplementation (before) followed by feeding period of twelve weeks. The withdrawal effect of the supplementation on milk yield was recorded for one week. DMI was recorded daily based on the amount of green fodder, wheat straw and concentrate offered and the residue left (Table 1). Body condition score was recorded based on fat cover in the brisket, on the ribs, back, hooks, pins and around the tail head adopting a 5-point scale method. Body weight was recorded at fortnightly intervals. Milk yield of individual buffalo was recorded daily during morning (6am) and evening (6pm) milking. Milk composition viz., fat, protein, lactose and SNF were estimated by Lactoscan machine. Blood samples were collected from rural buffaloes before feeding and at monthly interval on day 30, 60 and 90 of experiment. Plasma growth hormone (GH), insulin like growth factors (IGF-1), leptin, ghrelin, progesterone and estrogen levels were determined by enzyme specific enzyme immunoassay kits. Plasma glucose was determined by analytical kit and plasma NEFA was estimated by Shipe et al. (1980) method. Plasma triglyceride, total cholesterol and HDL were estimated by the commercially available analytical kits. Statistical analysis of data i.e. two ways ANOVA was carried out by sigma stat-3 programme. Effects were considered to be significant at probabilities ≤0.05 while a trend was assumed for probabilities between 0.05 and 0.1. The mean and standard errors are being presented in table and figure.
However, milk protein and lactose content was not influenced (p>0.05) by supplementation in both the groups. Solid not fat (SNF) was higher (p<0.01) in urban buffaloes than the rural buffaloes. Supplementation did not influence body condition score (BCS) of both the groups. The dry matter intake (DMI) increased (P<0.05) during the supplementation in rural and urban buffaloes. Body weight varied (P<0.05) between the groups. The body weight was not influenced by supplementation in rural buffaloes however a significant increase (p>0.05) in body weight of urban was observed (Table 2). Plasma GH level was numerically higher (p>0.05) before supplementation and was not influenced during the experimental period. Plasma leptin level was higher (p>0.05) before supplementation and a gradual decrease was observed during supplementation period (Table 3). However such decline in plasma ghrelin level was not observed. Plasma estrogen concentration was lower (p>0.05) before supplementation and increased upon supplementation during the experiment. Plasma progesterone level did not indicate any set pattern of change before and during supplementation periods. Plasma leptin, ghrelin and estradiol levels varied (p>0.05) between animal. Plasma IgG level was lower (p<0.05) before supplementation and increased during the supplementation period. Plasma IGF-1 level declined (p<0.01) during supplementation in comparison to before supplementation. Plasma glucose level increased (p<0.05) gradually during the supplementation period concomitant to increase in milk yield, however plasma NEFA levels declined gradually. Plasma NEFA varied (p>0.05) during different days of sampling. Further, plasma glucose and NEFA varied between animal (p>0.05). Plasma cholesterol and HDL increased during the supplementation period in comparison to control (Table 4). Plasma triglyceride also increased (p>0.05) steadily during the supplementation. Plasma urea and blood urea nitrogen levels were lower before supplementation and an increase (P<0.05) was observed during supplementation.  The urban buffaloes exhibited first post-partum heat earlier (65 day) in comparison to rural Murrah buffaloes (87day) in spite of similar number of artificial inseminations. Conception rate was higher (p<0.05) in urban than the rural Murrah buffaloes. The supplementation resulted in lesser service period in urban buffaloes by 51 days in comparison to rural buffaloes. The overall conception rate was 46.87 % in buffaloes. Supplementation generated additional income of Rs 115/day, however withdrawal of feeding declined (p<0.05) milk yield leading to loss of Rs 274.5/ buffalo/day. The cost: benefit ratio during the experiment was 1:5.

Discussion
The increases in milk production in both the groups of buffaloes suggest that the mixture of prilled fat was galactopoitic and sustained the milk production during the experiment. This was also evident from the significant decline in milk yield after the withdrawal of feeding. However inclusion of sweetener did not increased DMI though it was supposed to enhance the palatability of feed and increase in DMI. The greater response in milk yield and fat content in rural Murrah buffaloes indicated that energy was limiting the milk production. Such effects on milk yield have been reported earlier in mid-lactation cows fed with prilled fat 75g/d  and in buffaloes maintained in organized farm of the institute . The increase in milk fat content resulted probably due to elevated saturated fatty acids level in blood which is taken up by mammary gland for milk fat synthesis. The expected change in BCS was not observed in this study as increase in milk yield was persistent. Body condition score is used for accurate determination of energy reserves as energy balance during entire lactation period (Coffey et al., 2003). Due to this reason BCS have strongest genetic association with cow fertility (Banos and Coffey, 2010). The decline in plasma NEFA concentration which is an important energy marker and transporters of fatty acids in blood remain unaffected by prilled fat supplementation (Quiroz-Rocha et al., 2009). However in this study decline in plasma NEFA reflected less adipose tissue triacylglycerol mobilization (Pullen et al., 1989). Stressors and poor nutritional management during peripartum results in large increases in NEFA immediately after calving due to decrease in voluntary dry matter intake (Drackley, 1999). The enhanced milk production therefore could be attributed to prilled fat. The significant increase in DMI could be attributed to bypassing of the prilled fat and not affecting the rumen microflora. Cows in NEB have lower leptin concentrations, higher NEFA and produce more milk with lighter live weight compared to cows in positive EB (Liefers et al., 2003). Moreover plasma leptin concentration is positively correlated with glucose and insulin concentrations and negatively with plasma NEFA concentrations (Block et al., 2001). However such type of correlation and pattern was not evident as buffaloes were producing less milk yield than the cows. But in medium producer cows similar effect of prilled fat supplementation on plasma leptin concentration has been reported . The non-significant decline in plasma ghrelin was probably due to increasing stage of lactation and maintenance of milk yield in buffaloes. Ghrelin, a 28-amino acid octanoylated peptide is secreted primarily by cells in the abomasum in ruminants (Huang et al., 2006) and is the "ultimate anabolic hormone" because it causes the body to consume and store energy (Litwack et al., 2008). Singh et al. (2014) report no effect of prilled fat feeding on plasma ghrelin concentration in cows as observed in this study. Plasma GH did not vary significantly though its role as galactopoitic hormone in buffaloes have been established (Prasad and Singh 2010;Singh et al., 2014). GH partitions nutrient towards the mammary gland at the expense of other tissues (Aschenbach et al., 2011). An increase in plasma cholesterol, triglycerides was observed as expected but lower HDL level during supplementation suggest that animal health remains unaffected by prilled fat supplementation. The improved reproductive performance could be attributed to higher plasma cholesterol and higher IGF-I levels as both play important role in reproduction (Wadhwa et al., 2012). Plasma insulin, leptin and insulin-like growth factor-I control ovarian follicular development and serve as mediators of energy balance on cow's fertility (Diskin et al., 2003;Webb et al., 2004). The feeding supplement was highly economical for dairy farmers in urban and rural conditions and corroborates the similar findings on additional income generation in prilled fat fed buffaloes  and with calcium salts of fatty acids in cows (Naik et al., 2009;Parnerkar et al., 2011).

Conclusion
Supplementation of a mixture of prilled fat along with sweetener and toxin binder significantly improves milk yield and fat percent without affecting milk protein, lactose and body condition score. The supplementation increased plasma glucose, decreased NEFA and improves the reproductive performance. Further being cost effective it could be used successfully to augment the productive performance of rural and urban reared buffaloes.