Impact of dietary rumen-protected protein and fat supplementation on milk yield and composition in Murrah buffaloes

Introduction

The transition period, extending from approximately three weeks before calving to three weeks after calving, is recognized as one of the most critical phases in the productive life of dairy animals due to the profound physiological, metabolic, and endocrine changes that occur during this stage. Rapid fetal growth during late gestation, coupled with the onset of lactation, substantially increases nutrient requirements, which often exceed voluntary feed intake, resulting in a state of negative energy balance (NRC, 2001) [14] . This nutritional imbalance may adversely affect milk yield, milk composition, health status, reproductive performance, and overall productive efficiency. Buffaloes play a vital role in the dairy sector of many Asian countries, particularly India, where Murrah buffaloes are highly valued for their superior milk-producing ability, adaptability, and economic importance. Despite their high genetic potential, the productive performance of buffaloes is often limited by inadequate nutritional management during the transition period. Conventional feeding practices frequently fail to satisfy the increased energy and protein requirements associated with early lactation, leading to inefficient nutrient utilization and suboptimal productive performance.

In ruminants, a considerable proportion of dietary protein is degraded in the rumen, producing ammonia and resulting in the inefficient utilization of dietary nitrogen and associated energy losses. Similarly, supplementation with unprotected fats may impair rumen fermentation by reducing fiber digestion and altering microbial activity. Consequently, nutritional strategies that enhance the post-ruminal availability of nutrients without adversely affecting rumen function have received considerable attention. Among these, rumen protected protein and rumen protected fat have been extensively investigated for their potential to improve nutrient utilization and lactational performance. Rumen protected protein, commonly produced by formaldehyde treatment of oilseed cakes, minimizes ruminal protein degradation and increases the flow of undegraded dietary protein to the small intestine, thereby improving the availability of absorbable amino acids for productive functions (Chatterjee and Walli, 2003; Walli, 2005) [5,28] . Likewise, rumen protected fat provides a concentrated source of energy without interfering with normal rumen fermentation, thereby improving energy balance and supporting milk synthesis (Palmquist and Jenkins, 1980; Palmquist, 1991) [15,16] . Several studies have reported beneficial effects of supplementing rumen protected fat or rumen protected protein individually on milk yield and milk composition in dairy animals (Garg et al., 2003a; Ranjan et al., 2012; Sirohi et al., 2013) [9,18,22] . Furthermore, the combined supplementation of rumen protected protein and rumen protected fat has been shown to enhance lactational performance by improving nutrient availability and optimizing the synchronization of energy and protein utilization (Shelke et al., 2012b; Vahora et al., 2013; Katiyar et al., 2019; Mane et al., 2016) [20,26,11,12] . However, information regarding the combined effects of these nutritional interventions in transition Murrah buffaloes under Indian feeding conditions remains limited. Therefore, the present study was undertaken to evaluate the effects of dietary supplementation with rumen protected protein and rumen protected fat on milk yield and milk composition in Murrah buffaloes during the transition period.

Materials and Methods

Experimental site and duration

The experiment was conducted at the Dairy Farm of the Department of Animal Husbandry and Dairy Science, College of Agriculture, Pune, Maharashtra, India, from August to December 2016. The experimental period extended from 30 days before calving to 90 days after calving, during which all animals were maintained under standard farm management practices.

Experimental animals and design

Ten pregnant Murrah buffaloes were selected from the institute herd based on their Most Probable Producing Ability (MPPA) of approximately 1800 L milk yield. The animals were randomly allocated into two experimental groups, each comprising five buffaloes, and were maintained under identical housing and management conditions throughout the study. The control group (T 0 ) was fed a conventional basal ration, whereas the treatment group (T 1 ) received the basal ration supplemented with rumen protected protein and rumen protected fat.

Feeding treatments

The composition of the experimental diets offered to the animals is presented in Table 1. The concentrate mixture was provided twice daily according to the individual milk production of each buffalo throughout the experimental period.

Preparation of rumen protected protein

Groundnut cake was treated with 40% formaldehyde at the rate of 1.0 g formaldehyde per 100 g crude protein following the procedure described by Chatterjee and Walli (2003) [5] . Briefly, the required quantity of formaldehyde was diluted with water, uniformly sprayed onto the groundnut cake, and thoroughly mixed to ensure even distribution. The treated material was subsequently stored in airtight plastic bags for seven days before feeding to facilitate protein protection and stabilization.

Rumen protected fat supplementation

Commercial rumen protected fat (calcium salts of palm fatty acids) was procured from Berg and Schmidt Ingredients Pvt. Ltd., Pune, India. Buffaloes in the treatment group received rumen protected fat at the rate of 15 g per litre of milk produced throughout the experimental period. The supplement was thoroughly mixed with the concentrate mixture and offered during feeding.

Animal management

All experimental buffaloes were housed in a well-ventilated dairy shed with individual feeding facilities to prevent crossfeeding among animals. The animal shed was cleaned twice daily to maintain proper hygiene, and fresh drinking water was made available ad libitum throughout the experimental period.

Milk yield and milk composition

Individual milk yield was recorded daily by combining the morning and evening milkings throughout the study. Milk samples were collected at fortnightly intervals and analyzed using a pre-calibrated milk analyzer to determine milk fat, protein, lactose, solids-not-fat (SNF), and total solids (TS).

Chemical analysis

Representative samples of feed ingredients and concentrate mixtures were analyzed for dry matter (DM), crude protein (CP), ether extract (EE), crude fibre (CF), total ash (TA), and nitrogen-free extract (NFE) following the standard analytical procedures of the Association of Official Analytical Chemists (AOAC, 1995) [2] .

Statistical analysis

The experimental data were subjected to statistical analysis using an independent samples Student's t-test as described by Snedecor and Cochran (1995) [24] . Mean values of the two treatment groups (T 0 and T 1 ) were compared for all measured parameters. The results are expressed as mean ± standard error (SE), and differences between treatment means were considered statistically significant at P < 0.05. Individual animals were considered the experimental units for all statistical analyses.

Results and Discussion

Milk yield

Milk yield exhibited the typical lactation pattern in both experimental groups, with production increasing during early lactation and gradually declining towards the end of the experimental period. Throughout the study, however, buffaloes supplemented with rumen protected protein and rumen protected fat (T 1 ) consistently produced higher milk yield than those in the control group (T 0 ) (Table 2). The average daily milk yield was significantly (P < 0.05) greater in T 1 (7.62 ± 0.15 kg/day) than in T 0 (6.67 ± 0.21 kg/day), representing an increase of approximately 14.2%. The maximum milk yield was recorded during the third fortnight in T 1 (8.08 ± 0.68 kg/day), whereas the minimum yield was observed during the first fortnight in T 0 (6.36 ± 0.07 kg/day).

The improvement in milk yield may be attributed to the enhanced post-ruminal availability of nutrients. Formaldehyde treatment reduced the ruminal degradation of dietary protein and increased the flow of undegraded dietary protein to the small intestine, thereby improving the supply of absorbable amino acids for mammary gland function (Chatterjee and Walli, 2003; Walli, 2005) [5,28] . In addition, supplementation with rumen protected fat increased the energy density of the diet without adversely affecting rumen microbial fermentation, thereby providing additional energy to support milk synthesis (Palmquist and Jenkins, 1980; Palmquist, 1991) [15,16] . The combined supplementation of rumen protected protein and rumen protected fat likely improved the energy-protein balance during the transition period. Early lactation is characterized by increased nutrient demands accompanied by reduced voluntary feed intake, often leading to a negative energy balance. Enhancing the availability of absorbable amino acids together with additional energy substrates may have promoted more efficient nutrient partitioning towards milk synthesis, thereby improving overall lactational performance.

The findings of the present study are consistent with those of Bhosale et al. (2010) [3] , Sirohi et al. (2010) [23] , Ranjan et al.

(2012) [18] , Ramteke et al. (2014) [17] , Yadav et al. (2015) [29] , Sharma et al. (2016) [19] , and Desai et al. (2016) [6] , who reported significant improvements in milk yield following bypass fat supplementation. Likewise, Dosky et al. (2012) [8] , Sherasia et al. (2012) [21] , Vahora et al. (2012) [27] , Sirohi et al. (2013) [22] , and Amrutkar et al. (2014) [1] observed enhanced milk production in dairy animals supplemented with rumen protected protein. Similar positive responses to the combined supplementation of rumen protected protein and rumen protected fat have also been documented by Shelke et al. (2012b) [20] , Dhulipalla et al. (2013) [7] , Vahora et al.

(2013) 26 [] , Grewal et al. (2014) [10] , Mane et al. (2016) [12] and Katiyar et al. (2019) [11] .

Six percent fat-corrected milk (FCM)

Six percent fat-corrected milk (FCM) yield is considered a more reliable indicator of lactational performance because it accounts for variations in milk fat concentration. In the present study, the average 6% FCM yield was significantly (P < 0.05) higher in the supplemented group (T 1 ; 8.79 ± 0.17 kg/day) than in the control group (T 0 ; 7.32 ± 0.21 kg/day), representing an improvement of approximately 20.1%. The maximum 6% FCM yield was recorded during the sixth fortnight in T 1 (9.98 ± 0.67 kg/day), whereas the minimum value was observed during the first fortnight in T 0 (6.68 ± 0.06 kg/day). The higher FCM yield in the supplemented group can be attributed to the concurrent improvement in both milk yield and milk fat percentage.

The increased energy supply provided by rumen protected fat may have enhanced the availability of long-chain fatty acids for milk fat synthesis, whereas the greater availability of absorbable amino acids from rumen protected protein likely supported mammary gland metabolism and milk production. Furthermore, the synchronized supply of energy and protein is considered an important factor contributing to improved nutrient utilization and enhanced lactational efficiency in dairy animals.

The findings of the present study are in agreement with those of Ranjan et al. (2012) [18] , Ramteke et al. (2014) [17] , and Yadav et al. (2015) [29] , who reported increased FCM yield following bypass fat supplementation. Likewise, Vahora et al.

(2012) [27] , Sirohi et al. (2013) [22] , and Amrutkar et al. (2014) [1] observed similar improvements with rumen protected protein supplementation. Positive effects of the combined supplementation of rumen protected protein and rumen protected fat on FCM yield have also been reported by Shelke et al. (2012b) [20] , Dhulipalla et al. (2013) [7] , Vahora et al.

(2013) [26] and Mane et al. (2016) [12] .

Effect of dietary supplementation of rumen protected protein and fat on milk composition 3.3.1 Milk fat

Milk fat percentage was significantly (P < 0.01) higher in the supplemented group (T 1 ; 7.31%) than in the control group (T 0 ; 6.85%). The increase in milk fat content may be attributed to the greater intestinal availability of long-chain fatty acids supplied by rumen protected fat, which can be directly utilized for milk fat synthesis. Moreover, the enhanced post-ruminal availability of amino acids resulting from rumen protected protein supplementation may have improved nutrient utilization efficiency and supported mammary gland metabolism.

Approximately half of the fatty acids present in milk fat are synthesized from acetate and butyrate produced during ruminal fermentation, while a considerable proportion is derived from absorbed dietary fatty acids (Palmquist and Jenkins, 1980) [15] . Consequently, increasing the dietary supply of rumen protected fat can enhance milk fat concentration without adversely affecting normal rumen fermentation.

The results of the present study are consistent with those reported by Barley and Baghel (2009), Thakur and Shelke (2010) [20] , Sirohi et al. (2010) [23] , Sharma et al. (2016) [19] , and Mane et al. (2016) [12] , who observed significant improvements in milk fat percentage following bypass fat supplementation. Similar findings have also been reported with rumen protected protein supplementation by Dosky et al.

(2012) [8] , Sherasia et al. (2012) [21] , and Amrutkar et al.

Milk protein

Milk protein concentration was numerically higher in the supplemented group (T 1 ; 3.42%) than in the control group (T 0 ; 3.39%); however, the difference was not statistically significant. The lack of a significant treatment effect suggests that the additional supply of absorbable amino acids may have been preferentially utilized to support the increased milk yield rather than increasing the protein concentration of milk. The present findings are in agreement with those of Sirohi et al. (2013) [22] , Amrutkar et al. (2014) [1] , Grewal et al. (2014) [10] , and [25,13] .

Lactose

Milk lactose concentration was not significantly affected by dietary supplementation, with mean values of 5.22% and 5.29% recorded in T 0 and T 1 , respectively. Lactose is generally regarded as one of the most stable constituents of milk because its synthesis is largely regulated by mammary osmotic balance and is relatively insensitive to dietary manipulation. These findings are consistent with those reported by Sirohi et al. (2010) [23] , Ranjan et al. (2012) [18] , Sirohi et al. (2013) [22] , Singh et al. (2014), and Yadav et al. (2015) [29] , who likewise observed minimal effects of bypass nutrient supplementation on milk lactose concentration.

Solids-not-fat (SNF)

No significant differences were observed in solids-not-fat (SNF) content between the treatment groups. The mean SNF values remained within the normal physiological range for Murrah buffalo milk, indicating that supplementation with rumen protected protein and rumen protected fat enhanced milk production without adversely affecting milk quality. Similar observations have been reported by Sirohi et al.

(2013) [22] , Amrutkar et al. (2014) [1] , Shelke et al. (2011a) [20] , and Mane et al. (2016) [12] .

Total solids

Total solids content was numerically higher in the supplemented group (T 1 ; 17.59%) than in the control group (T 0 ; 17.25%), although the difference was not statistically significant. The slightly higher total solids content may be attributed to the increase in milk fat percentage observed in the supplemented animals. Murrah buffalo milk is naturally characterized by a high total solids content, which contributes to its excellent processing characteristics and suitability for the manufacture of valueadded dairy products. Recent studies have highlighted the importance of nutritional interventions that improve milk production while maintaining the compositional stability and processing quality of buffalo milk.

Conclusion

The present study demonstrated that dietary supplementation with rumen protected protein and rumen protected fat during the transition period positively influenced the lactational performance of Murrah buffaloes. Buffaloes receiving the supplemented diet produced significantly higher milk yield, 6% fat-corrected milk (FCM) yield, and milk fat percentage than those fed the conventional diet. In contrast, milk protein, lactose, solids-not-fat (SNF), and total solids were not significantly affected, indicating that the supplementation enhanced milk production without compromising the overall compositional quality of milk.

The improvement in productive performance may be attributed to the greater post-ruminal availability of absorbable amino acids from rumen protected protein and the increased energy supply provided by rumen protected fat. The synchronized availability of protein and energy during early lactation likely improved nutrient utilization efficiency, alleviated the negative energy balance commonly experienced during the transition period, and promoted greater nutrient partitioning towards milk synthesis. These nutritional advantages contributed to enhanced lactational performance while maintaining normal milk composition. Overall, the findings indicate that strategic supplementation with rumen protected protein and rumen protected fat is an effective nutritional approach for improving milk production in transition Murrah buffaloes. The combined supplementation not only enhanced milk yield and milk fat content but also maintained desirable milk quality characteristics, highlighting its practical value under tropical production conditions. Adoption of this feeding strategy may therefore contribute to improved productivity and profitability of buffalo dairy farming, particularly during the nutritionally demanding transition period. Further studies involving larger animal populations and different stages of lactation are recommended to validate these findings and to optimize supplementation strategies under diverse management and feeding systems.

Acknowledgement

AcknowledgementsThe authors gratefully acknowledge the Department of

References

1. Amrutkar SA, Thakur SS, Pawar SP. Economics of supplementing rumen protected methionine and lysine in the ration of lactating crossbred cows. Indian J Anim Nutr. 2014;31(1):14-19.
2. Association of Official Analytical Chemists. Official methods of analysis. 16 th Ed. Arlington (VA): AOAC International; 1995.
3. Bhosale AB, Gadegaonkar GM, Patil MB, Kank VD, Hol BG, Gulavane SU. Effect of bypass fat supplementation on fat corrected milk yield and feed efficiency in lactating buffaloes. In: Proceedings of the International Buffalo Conference; 2010; New Delhi, India. p. 111.
4. Cavallini D, Lamanna M, Colleluori R, Silvestrelli S, Ghiaccio F, Buonaiuto G, et al. The use of rumenprotected amino acids and fibrous by-products can increase the sustainability of milk production. Front Vet Sci. 2025;12:1588425.
5. Chatterjee A, Walli TK. Effect of formaldehyde treatment on effective protein degradability and in vitro post-ruminal digestibility of mustard cake. Indian J Anim Nutr. 2003;20(2):143-148.
6. Desai VR, Padheriya YD, Parnerkar S. Effect of feeding bypass fat on milk production and economics of feeding in buffaloes of tribal areas of Panchmahal and Vadodara. Indian Vet J. 2016;93(3):66-68.
7. Dhulipalla SK, Venkateswarlu S, Raja KK. Effect of supplementation of Milk Dhara on milk yield and milk composition in graded Murrah buffaloes. J Adv Vet Res. 2013;3:89-92.
8. Dosky KNS, Jaaf SSA, Mohammed LT. Effect of protected soybean meal on milk yield and composition in local Meriz goats. Mesopotamia J Agric. 2012;40(1):110.
9. Garg MR, Sherasia PL, Bhanderi BM, Gulati SK, Scott TW. Effect of feeding rumen protected protein on milk production in lactating buffaloes. Anim Nutr Feed Technol. 2003;3:151-157.
10. Grewal RS, Tyagi N, Lamba JS, Ahuja CS, Saijpaul S. Effect of bypass fat and niacin supplementation on productive performance and blood profile of lactating crossbred cows under field conditions. Anim Nutr Feed Technol. 2014;14:573-581.
11. Katiyar GS, Vishal M, Sharma RK, Anurag B, SK, Andonissamy J, et al. Effect of rumen-protected nutrients on feed intake, body weights, milk yield, and composition in Murrah buffaloes during early lactation. Trop Anim Health Prod. 2019;51(8):2297-2304.
12. Mane SH, Fulpagare YG, Deokar DK, Kankhare DH, Adangle SA. Milk yield and composition in crossbred cows as influenced by feeding rumen protected protein and fat. Indian J Anim Nutr. 2016;33(1):114-117.
13. Muruz H, Çelik S, Selçuk Z, Bölükbaş B, Ertan S. Early lactational response to rumen-protected methionine supplementation in dairy buffalo fed forage-based diets. S Afr J Anim Sci. 2024;54(1):77-85.
14. National Research Council. Nutrient requirements of dairy cattle. 7 th Rev. Ed. Washington (DC): National Academy Press; 2001.
15. Palmquist DL, Jenkins TC. Fat in lactation rations: Review. J Dairy Sci. 1980;63(1):1-14.
16. Palmquist DL. Influence of source and amount of dietary fat on digestibility in lactating cows. J Dairy Sci. 1991;74:1354-1360.
17. Ramteke PV, Patel DC, Parnerkar S, Shankhpal SS, Patel GR, Katole SB, et al. Effect of feeding bypass fat prepartum and during early lactation on productive performance in buffaloes. Livest Res Int. 2014;2(3):6367.
18. Ranjan A, Sahoo B, Singh VK, Srivastava S, Singh SP, Pattanaik AK. Effect of bypass fat supplementation on productive performance and blood biochemical profile in lactating Murrah buffaloes. Trop Anim Health Prod. 2012;44(7):1615-1621.
19. Sharma S, Singh M, Roy AK, Thakur S. Effect of prepartum prilled fat supplementation on feed intake, energy balance and milk production in Murrah buffaloes. Vet World. 2016;9(3):256-259.
20. Shelke SK, Thakur SS, Amrutkar SA. Effect of feeding protected fat and proteins on milk production, composition and nutrient utilization in Murrah buffaloes (Bubalus bubalis). Anim Feed Sci Technol. 2012;171:98107.
21. Sherasia PL, Garg MR, Bhanderi BM. Effect of feeding slow ammonia release and protected protein supplement in lactating cows and buffaloes. Indian J Anim Res. 2012;46(2):168-171.
22. Sirohi SK, Walli TK, Garg MR, Kumar B. Effect of formaldehyde treated mustard cake on nutrient utilization and milk production performance in crossbred cows fed wheat straw based diet. Indian J Anim Nutr. 2013;30(1):5-11.
23. Sirohi SK, Walli TK, Mohanta RK, Sahu NP. Effect of bypass fat supplementation on production performance in dairy animals. Indian J Anim Nutr. 2010;27(1):1-8.
24. Snedecor GW, Cochran WG. Statistical methods. 8 th Ed. Ames (IA): Iowa State University Press; 1995.
25. Sun CH, Lee JS, Nejad JG, Kim WS, Lee HG. Effect of a rumen-protected microencapsulated supplement from linseed oil on the growth performance, meat quality, and fatty acid composition in Korean native steers. Animals. 2021;11(5):1253.
26. Vahora SG, Parnerkar S. Effect of feeding bypass nutrients on milk production and composition in buffaloes under field conditions. Indian J Anim Nutr. 2013;30(1):67-71.
27. Vahora SG, Kore KB, Parnerkar S. Feeding of formaldehyde-treated protein meals to lactating buffaloes: Effect on milk yield and composition. Livest Res Rural Dev. 2012;24:2.
28. Walli TK. Bypass protein technology and the impact of feeding bypass protein to dairy animals in tropics: A review. Indian J Anim Sci. 2005;75(1):135-142.
29. Yadav G, Roy A, Singh M. Effect of prilled fat supplementation on milk production performance of crossbred cows. Indian J Anim Nutr. 2015;32(2):133138.