MILK COMPOSITION AND PRODUCTIVITY OF HOLSTEIN COWS IN RYEGRASS GRAZING AND CRUDE GLYCERIN IN THE DIET

Objective: The aim of this project was to evaluate the effect of crude glycerin addition in the diet, milk composition and milk yield in dairy milking cows on different stages of lactation. Theoretical framework: It is necessary to balance the supply of protein and energy in the diet of lactating cows. Within this context, a by-product of the Biodiesel industry, called crude glycerin, is low cost and has a high energy value for ruminants, since its main component is glycerol, which ruminants have the capacity to use as a precursor gluconeogenic (Chung et al., 2007). Methods: The experiment was performed with Holstein cows (n=18), divided in two groups of treatment: 1) Basal diet without crude glycerin addition, 2) Diet with 10% of crude glycerin addition in the dry matter. Individual milk samples were collected for the analysis of the percentage of Fat, Protein, Lactose, Total Solids and Somatic Cell Count (Cs mL-1), as well as milk production (liters -1 and corrected for 4% fat). Results and conclusion: It was concluded that the addition of crude glycerin in the diet had


INTRODUCTION
Sustainable development is defined as the result of awareness of the global links between environmental problems, socio economic issues related to social inequality and concerns about a healthy future for humanity.Given this, the Brazilian Agenda 21 idealizes sustainability, permeating all dimensions of life: economic, social, cultural, territorial, scientific and technological, political and cultural, linking the government, the productive sector and society (Sontag et al., 2016).
In the Brazilian context, there have been studies related to sustainability and supply chains in both theoretical and practical contexts.However, research on the topic is growing, which suggests a great opportunity for the development of new studies in the country (Alves et al., 2018).Given this, crude glycerin comes as an alternative that favors the use of a byproduct of biodiesel production, which is clean energy.
The increase of milk production in volume and composition is dependent on the use of forage of excellent nutritional value (Ribeiro Filho et al., 2009).In this context, annual ryegrass (Lolium multiflorum Lam.), High protein forage, has been widely used during the cold season in the Southern Region of Brazil.
However, it should be noted that for lactating cows, it is necessary to balance the supply of protein and energy in the diet.Thus, interest in the inclusion of energetic byproducts in the diet of lactating cows has increased, focusing on this balance, using low-cost alternative sources, especially when we subject the animals to grazing on grasses such as ryegrass, which is commonly seen in southern Brazil.
Within this context, a by-product of the Biodiesel industry, called crude glycerin, is low cost and has a high energy value for ruminants, since its main component is glycerol, which ruminants have the capacity to use as a precursor gluconeogenic (Chung et al., 2007).
Due to the lack of data showing the effect of the inclusion of crude glycerin on the milk composition and yield this project was performed.The objective of this study was to evaluate the effect of the inclusion of crude glycerin in the dry matter of the total diet, of lactating cows in grazing on ryegrass.

THEORETICAL FRAMEWORK
According to Krehbiel (2008), most of the glycerol contained in the crude glycerin is fermented by ruminal microorganisms, providing high levels of volatile fatty acids in the rumen, mainly propionate and butyrate, which in turn are metabolized in the liver to oxaloacetate, through the Krebs cycle, and can be used to form glucose through the gluconeogenic route.Only a small amount of glycerol is absorbed directly by the rumen wall, in which case glycerol is metabolized in the liver and then directed to gluconeogenesis by the action of the glycerol kinase enzyme, so converts it to glucose (Boyd et al., 2009).
Due to this increase in the energy supply of glucose production, the inclusion of crude glycerin also has the potential to modify milk composition, since glucose reaches milk secreting cells in the mammary gland.Most (60-70%) are used in the synthesis of lactose, and the remainder stimulates protein synthesis, or can be used as a source of glycerol for fat formation and as a precursor in fat synthesis.The secretion of lactose in the alveolar lumen causes water to enter, exerting important control on the volume of milk.

MATERIAL AND METHODS
The research protocol (n.09 / 2014, opinion: 019/2014) followed the guidelines recommended by the Committee on Ethics in Animal Use.During the period from July to August of 2015, the experimental trial was conducted in the Augusto Pestana city in the state of Rio Grande do Sul,Brazil (Latitude: 28 ° 31 '2' 'South,Longitude: 53 ° 59' 39 '' West,Elevation: 385m).
Holstein lactating dairy cows (n=18) between 600 ± 50 kg body weight (BW) and >60 days in milk (DIM) were divided among 2 treatments groups: 1) Basal diet, without addition of crude glycerin, 2) Diet with addition of 10% crude glycerin in the total dry matter (DM) of the diet.The experimental period lasted 56 days, divided into four periods of 14 days, and in the first experimental period all cows received the same diet for standardization (called the adaptation period).Subsequently, it was randomly assigned to cows in pairs, by DIM (from 60 to 120 days, from 121 to 200 days and from 201 to 320 days) and similar milk production in a sequence of three periods (1, 2 and 3), in simple reversion (Cross-over).
At the beginning of the adaptation period all the animals were evaluated and only healthy animals on clinical and gynecological examination were included in the experiment.
The cows were maintained in grazing of reygrass (Lolium multiflorum Lam.), Bar HQ®, tetraploid, under rotational grazing, in a single herd in the experimental area, managed in order to provide a minimum of dry matter supply of 25 kg / animal /day.
Cows from each treatment were submitted to the same management and feeding conditions, ryegrass grazing in the interval between milking and supply of concentrate (according to milk production) plus the supply of 10 kg of corn silage per day, differing only between treatments the inclusion of 10% or not of crude glycerin.All animals were weighed at the beginning of each experimental period to determine daily crude glycerin consumption based on 3% dry matter intake by live weight.
The concentrate used was commercially pelleted with 17% of crude protein, which contained soybean meal as protein source, ground corn and rice bran as source of energy foods in its basic composition.The amount of concentrate supplied to each animal was changed whenever necessary and followed, as a practical criterion, the supply of 1kg of concentrate for every 5 liters of milk produced per day cow.The animals were mechanically milked twice a day at 7:00 a.m. and 5:00 p.m., and received the feed in individual head locks, with separate troughs, shortly after milking in the morning and before milking in the afternoon.During the remaining time, the animals remained in pasture areas with free access to drinking water.
Forage samples were collected by grazing simulation at the entrance of each pen during the experiment, and a composite sample was then performed for each period.The samples were kept frozen until they were sent to the laboratory for analysis.Samples of corn silage and concentrate were collected at each period as well.The dry matter, mineral matter, organic matter, crude protein, neutral detergent fiber, acid detergent fiber, ethereal extract and crude fiber contents of all diet components were evaluated in the food samples, Table 1.The analysis of the diet for each experiment period with and without crude glycerin is detailed in Table 2.The green material was dried in an oven with forced air circulation (55 °C) to constant weight, milled (1 mm sieve) and stored for further analysis.The dry matter (DM) contents of the food samples were determined by oven drying at 105ºC to constant weight.The ash content was determined by combustion at 600°C for 4 hours and organic matter (OM) by mass difference.The total nitrogen (N) of the food samples was determined by the Kjeldahl method (METHOD 984.13;AOAC, 1997) and determined crude protein and crude fiber.Neutral detergent fiber (NDF) analysis was based on procedures described by Mertens (2002) using thermostable α-amylase, except for those weighed into polyester filter bags (porosity 16 μm) and treated with detergent neutral in autoclave at 110° C for 40 minutes (Senger et al., 2008).Concentrations of acid detergent fiber (ADF) in acid detergent (AD) were analyzed according to AOAC Method 973.18 (1997), except for those that were weighed into polyester filter bags (16 μm porosity) and treated with autoclave acid detergent at 110° C for 40 minutes (Senger et al., 2008).Ethereal extract (EE) contents were obtained by extraction with ethyl ether in a reflux system at 180° C for 2 hours.After feeding in the canzil the leftover food was weighed and the data compiled, forming an average of leftovers at the end of each period, per animal.
Two days at the end of each experimental period, an aliquot (approximately 40 mL) of milk from each animal of both milking (morning and afternoon) was collected and packaged in a bottle containing Bronopol preservative (2-bromo-2-nitro-3-propanediol).The flasks with the samples were identified with the number of the animal and sent to the Official Laboratory, for the purpose of analyzing the percentage of Fat, Protein, Lactose, Total Solids and Somatic Cell Count (Cs mL mL -1 ).As the milk composition was analyzed by the infrared method and the somatic cell count by flow cytometry.
The milk production (liters day -1 ) was analyzed in the last six days of each experimental period, by means of the measurement of milk and milk production by means of milk weighing through a semi-automated milking system, after an average per experimental period.Milk production was also analyzed and corrected for 4% fat production, according to the formula: [(0,4) x (milk production) + 15 (milk production x% fat / 100)] (NRC, 2001).
The variables expressed as percentages (fat, protein, lactose and total solids) were transformed by the application of square root arcsine of their percentage values and later analyzed (Markus, 1973).The somatic cell count values were transformed by application of the logarithm base 10 and subsequently analyzed (Ng- Kwai-Hang et al., 1982).Although the statistical analysis was done with transformed values, their original values are presented in the tables that follow, to facilitate interpretation.
The results concerning the composition and milk production variables were analyzed using the MIXED procedure of SAS (Statistical Analysis System, version 9.2., 2000).The inclusion of crude glycerin was considered as a fixed effect and the block as a random effect (blocking days in lactation).The results of each animal were incorporated into the mean of the groups (treated or not).The data were submitted to analysis of variance and the treatment effect was evaluated by the Tukey test for paired data, the contrasts were considered significant when the P value was <0.05 and P <0.10.

RESULTS
After a milk composition analyses, according period of lactation a significant increase in fat (P = 0.0024), protein (P = 0.0242), total solids (P = 0.0164) and somatic cell count (P = 0.0017) was found, proportionally of the increase of DIM (Table 3).The milk production in liters (P = 0.0001) and the fixed production to 4% of fat (P = 0.0008) of milk had a significant effect with higher values at the beginning of lactation, Table 3.The lactose content in milk was not influenced by the lactation period (P = 0.3611, Table 3).The highest production of milk in liters day-1 and corrected to 4% of fat occurred at the beginning of lactation, Table 3.The inclusion of 10% crude glycerin in the diet had no significant effect on any variable response evaluated in relation to milk composition or milk yield (Table 4).Only for somatic cell counts there was a tendency (P = 0.0640) to increase when crude glycerin was included in the animals diet.

RESULTS AND DISCUSSION
Among the milk's component, the fat content is what can be more easily varied depending on the feed, generally decreasing with the increase in the volume of production, which was verified in this study.While the fat may vary from 2 to 3 percentage units, the milk protein content has a much lower range of variation, ranging from 0.3 to 0.4% (Wittwer, 2000).In this case, it was observed in the same way, according to data presented.
The significant difference observed in total solids on the different days of lactation was expected, considering the effect on milk fat and protein found.Changes in fat content can inform on rumen fermentation, cow health conditions and food management.According to NRC (2001), in an abnormal fermentation situation, due to the excess of polyunsaturated acids in the diet, enzymatic activities in the udder are reduced, reducing the synthesis of fatty acids with less than 16 carbons, the acid acetic as the main precursor, and consequently reduces the 8 milk fat content.Which was predictable since lactose is the component of milk that is less altered in view of its important osmotic role in milk (Fonseca & Santos, 2000).
The linear increase in milk production was consistent with the reduction in days of lactation, but the lactation days did not affect lactose production, but there was influence of lactation days on milk somatic cell count, which increased with rise of the days in milk period.These results were predictable since, at the end of lactation, there was an increase in CCS, due to a higher natural desquamation of the mammary gland epithelium (Monardes, 1994).
The increase of the milk protein simultaneously to the increase of somatic cells verified in this study was also observed by Noro et al. (2006).This increase is due not only to the cellular protein, but also to the change in the permeability of the mammary alveolar membrane, leading to increased influx of lactalbumin and immunoglobulins into the gland (Pereira et al., 1999).Concomitant to the increase in milk serum proteins, casein decreases, due to its degradation by bacterial and leukocyte proteases and by the decrease of its synthesis, which is an undesirable effect (Fonseca & Santos, 2000).Ribas et al. (2003) observed that milk protein behaves inversely to milk production during lactation, with gradual increase until the end of lactation, corroborating the data of this study.
The decrease in production at the end of lactation coincides with the increase of somatic cells, which indicates that cows with inflammatory processes mammals suffer significant reduction in production, coinciding with data reported by Noro et al. (2006).
In relation to the inclusion of crude glycerin in the diet, ruminants have the ability to use glycerol, the main component of glycerin, as a gluconeogenic precursor (Chung et al., 2007) for the maintenance of plasma glucose levels, since a large part of glycerol , which is contained in the diet undergoes fermentation by ruminal microorganisms, providing high levels of volatile fatty acid production in the rumen, mainly propionate and butyrate, which will be used as main energy sources by the animal (Boyd et al.When absorbed directly by the wall of the rumen, glycerol is metabolized in the liver and directed to gluconeogenesis by the action of the enzyme glycerol kinase, which converts it to glucose.Part of the glycerol can be fermented to propionate in the rumen, which in turn is metabolized to oxaloacetate via the Krebs cycle in the liver and can be used to form glucose through the gluconeogenic route (Krehbiel, 2008).
In spite of presenting this role, in this study the inclusion of 10% crude glycerin in the diet had no significant effect on any variable response evaluated in relation to milk composition or milk yield (Table 4).Only for somatic cell counts there was a tendency to increase when crude glycerin was included in the animals diet, but for this variable the coefficient of variation of the analysis was high, suggesting that more studies should be conducted to clarify this effect, Table 4.
Studies by Fisher et al. (1973);DeFrain et al. (2004) and Ogborn (2006) also found no significant effects of glycerin inclusion on milk composition or milk yield.DeFrain et al. ( 2004) reported tendencies for lower fat production in milk when crude glycerin was included, this trend did not occur in this study (Table 4).Donkin et al. (2009), testing the treatments 0, 5, 10 and 15% inclusion of glycerin in the dry matter of the diets, providing corn silage as bulky, stated that glycerol is a suitable substitute for corn grain in diets for dairy herd and which may be included in feeding stuffs up to 15% dry matter, without adverse effects on milk production or milk composition.This evidence is in agreement with the one found in this study, in which there was no reduction in milk production or alteration in its composition, since glycerol inclusion was similar (10% in DM) although we evaluated in grazing conditions to the crude glycerin diet.Khalili et al. (1997);DeFrain et al. (2004); Bodarski et al. (2005) tested the inclusion of lower levels of glycerol in DM and also found no significant difference in milk production.

Table 1 -
Results of the bromatological analyzes of the three experimental periods, for ryegrass pasture, corn silage and concentrate.

Table 2 -
Results of the analysis for nutritional composition of the diet of the experimental periods (% DM).

Table 3 -
Average values of fat, protein, lactose, total solids, somatic cell count and milk production, of Holstein cows, in different periods of lactation.

Table 4 -
Average values of fat, protein and lactose, somatic cell count and milk production, in different lactation periods, of Holstein cows treated with a additional diet or without 10% crude glycerin in total dry matter.Milk Composition and Productivity of Holstein Cows in Ryegrass Grazing and Crude Glycerin in The Diet ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.2 | p.1-11 | e03635 | 2024.* Different letters on the same line are significant for P <0.05 and tendency for P <0.10, Test Tukey.Source: Prepared by the authors