Dairy ruminants commonly undergo peripartum dry matter intake (DMI) depression that can affect the lactation. Reduced DMI affects mammary glucose supply and milk production (Brown and Allen, Reference Brown and Allen2013; Wankhade et al., Reference Wankhade, Manimaran, Kumaresan, Jeyakumar, Ramesha, Sejian, Rajendran and Varghese2017). Thus, nutritional strategies have been devised to overcome this problem (Otaru et al., Reference Otaru, Adamu, Ehoche and Makun2011; Abd-Allah, Reference Abd-Allah2013; Alba et al., Reference Alba, Favaretto, Marcon, Saldanha, Leal, Campigoto, Souza, Baldissera, Bianchi, Vedovatto and Da Silva2020). In many cases, such strategies involve using by-product feedstuffs with special traits that could help to improve DMI.
Crude glycerin (CG) is a by-product of the biofuel industry that contains a high glycerol concentration (about 800 g/kg) with glycogenic properties. Thus, glycerin has been studied (Ribeiro et al., Reference Ribeiro, Carvalho, Silva, Costa, Bezerra, Cambuí, Barbosa and Oliveira2018; Porcu et al., Reference Porcu, Sotgiu, Pasciu, Cappai, Barbero-Fernández, Gonzalez-Bulnes, Dattena, Gallus, Molle and Berlinguer2020; Larson et al., Reference Larson, Jaderborg, Paulus-Compart, Crawford and DiCostanzo2023), especially as a corn substitute, in ruminant and nonruminant animals (Madrid et al., Reference Madrid, Villodre, Valera, Orengo, Martínez, López, Megías and Hernández2013; Hales et al., Reference Hales, Foote, Brown-Brandl and Freetly2015). Glycerol may have three major fates inside the rumen, namely ruminal escape, absorption through the ruminal wall or fermentation to short-chain fatty acids. Previous studies have shown that glycerol increases the ruminal synthesis of propionate and butyrate over acetate (Ferraro et al., Reference Ferraro, Mendoza, Miranda and Guti??rrez2009; Wang et al., Reference Wang, Liu, Huo, Yang, Dong, Huang and Guo2009; El-Nor et al., Reference El-Nor S, AbuGhazaleh, Potu, Hastings and Khattab2010). As acetate is related to methane production in the rumen, glycerol has been reported to reduce in vitro methane production and increase the efficiency of dietary energy use in ruminants (Lee et al., Reference Lee, Lee, Cho, Kam, Lee, Kim and Seo2011). Even when it escapes the rumen along with digesta or is absorbed through the ruminal wall, glycerol has an energy-related metabolic fate, especially as a substrate for gluconeogenesis.
Despite having a different ruminal fermentation compared to cereal grains (rich in starch), CG has been reported to replace corn grain (up to 10% DMI) in diets for dairy cows without affecting the efficiency of milk production (Bruni et al., Reference Bruni, Carriquiry, Delgado and Chilibroste2017). Polizel et al. (Reference Polizel, Susin, Gentil, Ferreira, de Souza, Freire, Pires A, Ferraz, Rodrigues and Eastridge2017) reported no effects of CG on DMI and milk production in ewes (in late pregnancy and early lactation) fed up to 100 g CG/kg DM.
CG is usually fed as part of the ration. However, drinking water may be useful for administering nutrients to animals (Osborne et al., Reference Osborne, Radhakrishnan, Odongo, Hill and McBride2008). Osborne et al. (Reference Osborne, Odongo, Cant, Swanson and McBride2009) supplemented dairy cows with glycerol in drinking water (20 g/l) and reported lower DMI and serum 3-hydroxybutyrate. However, no effects on milk production were observed in this study. Sá et al. (Reference Sá, Borges, Macedo Junior, Santos, Cavalcanti, Alvarenga, Martins and Campolina2017) supplemented transition dairy ewes with CG and reported that CG did not negatively affect milk production or milk composition. To our knowledge, there are no studies performed to evaluate the effects of CG supplementation in drinking water in dairy ewes throughout the entire lactation period. We hypothesize that CG supplementation diluted in drinking water to dairy ewes could be a good feeding practice to supply dietary energy and support milk production throughout the lactation period. Thus, this study was performed to determine the effects of CG supplementation in drinking water on DM and nutrient intake, milk production and milk composition in dairy ewes.
Material and methods
The guidelines of the Local Ethical Committee for the Use of Animals in Experimentation at Universidade Federal de Uberlândia (Uberlândia, Brazil) were followed, and all experimental procedures were approved (CEUA, protocol 056/11)
Animals, diets and experimental procedures
Twenty multiparous Lacaune × East Friesian ewes (62.68 ± 5.95 kg BW) carrying a single lamb were used. The animals were assigned to four dietary treatments in a complete randomized design from late gestation extending throughout the entire lactation (119 d). Treatments consisted of doses of CG supplementation in drinking water as follows: (1) no CG supplementation, (2) 15.0 g CG/kg DM, (3) 30.0 g CG/kg DM and (4) 45.0 g CG/kg DM. CG contained 854.0 g/kg DM and, on a per kilogram DM basis, 0.6 g of crude protein, 57.1 g of ash, 134.1 g of ether extract, 807.0 g of glycerol and 3954 kcal of crude energy. CG was supplemented based on the expected DM intake and offered mixed in 10 l of water to provide the desired experimental dose. The water was supplied in individual buckets, and the intake was recorded daily.
The ewes were weighed on the first day of the experimental period and then every 15 d. The ewes were housed in individual pens with free access to water and were fed twice a day (8:00 h and 17:00 h) a diet containing 503.3 g/kg Tifton hay and 496.7 g/kg concentrate (online Supplementary Table S1). The diet was formulated to meet the requirements of lactating ewes (NRC, 2007) fed for ad libitum intake.
Milk production was determined from day 11 to 119 of lactation in two different ways. From day 11 to 45, lambs were kept with their respective dams from 19:00 to 07:00 h, then the milk production was estimated by adding the milking record (15:00 h) with the estimate of daily lamb milk intake, which was obtained after weighing lambs at 19:00 and 07:00 h (urine and feces excretion were not accounted for). From day 46 to 119 postpartum, as lambs had no more access to ewes, the milk production was determined via milking (15:00 h). Milk samples (40 ml) were taken every seven days and stored at 4°C with a preservative (2-bromo-2-nitropropam1,3 diol) until analysis of composition (fat, protein, and lactose). Data on milk production were presented as standardized milk yield (6.5% fat and 5.8% protein), as previously reported by Pulina and Nudda (Reference Pulina and Nudda2004).
Blood samples were collected every 15 d from the jugular vein before (0 h) and 3, 6, and 9 h after the morning meal using fluoride-containing tubes. Serum was separated from blood by centrifugation at 5000 rpm for five min at room temperature and stored at −18°C until glucose analysis.
Chemical analysis
Forage, concentrate and fecal samples were ground through a Wiley mill equipped with a 1 mm screen for analysis. Dry matter and crude protein analyses were performed according to AOAC (2000) techniques (method 934.01 and 984.13, respectively). The concentrations of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed using the sequential method (using α-amylase, urea, and sodium sulfite) as previously suggested by Van Soest et al. (Reference Van Soest, Robertson and Lewis1991).
Serum glucose was determined in an automated biochemical analyzer using a commercial kit (Labtest Diagnóstica S.A., Lagoa Santa, Brazil). Milk composition was analyzed by infrared spectrophotometry (Bentley model 2000; Bentley Instrument Inc., Chaska, MN, USA).
Statistical analysis
Data were analyzed using version 9.1 of the Statistics and Genetics Analysis System (SAEG). Data were submitted to analysis of variance with the following split-plot model:
$$Y_{{\rm ijk}}\,{\rm} = {\rm \mu }\,{\rm} + X_{\rm i}\,{\rm} + {\rm \sigma }_{{\rm j( i) }}\,{\rm} + {\rm \beta }_{\rm j}\,{\rm} + {\rm \tau }_{\rm k}\,{\rm} + {\rm \varepsilon }_{{\rm ijk}}$$where: Yijk = dependent variable, μ = overall mean; X i = effect of treatment (i = 0, 15.0, 30.0, and 45.0 g CG/kg DM); σj(i) = effect of weeks of lactation; βj = effect of animal (j = 1 to 5); τk = interaction between treatment and lactation week; Ɛijk = random residual error. Orthogonal contrasts and polynomial regression were used to test the linear and quadratic effects of CG doses. Significance was declared at P ≤ 0.05, unless otherwise stated.
Results
No interaction (P > 0.05) was observed between treatment and lactation week for DM and nutrient (CP, NDF and ADF) intake (Table 1). DM and nutrient intake were linearly reduced (P < 0.01) with CG supplementation. DMI was quadratically (P < 0.05) affected by the lactation phase, expressed as weeks of lactation (Table 1 and Fig. 1). The maximum DMI was reached at week 8 and averaged 1.83 ± 0.39 kg d−1 (mean ± sd).
Fig. 1. Regression equations of dry matter intake (DMI) of ewes supplemented with different levels (0, 15.0, 30.0, and 45.0 g/kg DM) of crude glycerin diluted in water and according to the lactation week.
Table 1. Nutrient intake and serum glucose of ewes supplemented with different levels of crude glycerin diluted in water
a Coefficient of variation, ** P < 0.01, *P ≤ 0.05, ns, not significant.
CG linearly reduced (P < 0.05) water intake expressed as kg d−1 (Table 1). However, it was not affected when expressed as a percentage of body weight or metabolic BW. The water to DM ratio was linearly increased (P < 0.01) with CG supplementation.
There was no interaction between experimental doses of CG and sampling hour (0, 3, 6, and 9 h post-feeding) for serum glucose (data not shown). Therefore, only mean values are presented for the four blood sampling times (Table 1). No effect of CG doses on serum glucose was observed.
There was no interaction between treatment and week of lactation for milk yield and composition. Standardized milk production decreased linearly (P < 0.05) with crude glycerin experimental doses (Table 2 and Fig. 2). The lactose concentration in milk was increased linearly (P < 0.01) with experimental doses of CG. Protein and urea concentrations in milk were increased quadratically (P < 0.01) with experimental doses of CG where greater values were observed with 15.0 g CG/kg DM (Table 2 and Fig. 3). No treatment effect was observed for fat concentration or yield. Protein, fat, and lactose yields were linearly reduced (P < 0.05) with experimental doses of CG.
Fig. 2. Regression equations of milk yield (standardized to 6.5% of fat and 5.8% of protein) and yield of fat, protein and lactose in milk of ewes supplemented with different levels (0, 15.0, 30.0, and 45.0 g/kg DM) of crude glycerin diluted in water and according to the lactation week.
Fig. 3. Regression equations of milk fat, protein and lactose percentage of ewes supplemented with different levels (0, 15.0, 30.0, and 45.0 g/kg DM) of crude glycerin diluted in water and according to the lactation week.
Table 2. Body weight changes, milk production and composition, and feed conversion of ewes supplemented with different levels of crude glycerin diluted in water
a Standardized to 6.5% of fat and 5.8% of protein.
b Feed conversion (DMI (g)/milk yield (g).
cN-efficiency = (milk N (g d−1) × 100/N intake (g d−1)). ** P < 0.01, *P ≤ 0.05, ns, not significant.
The feed conversion during the overall lactation and the after-weaning period was not influenced by the experimental doses of CG (Table 2). However, CG supplementation in drinking water had a quadratic effect on feed conversion during pre-weaning (P < 0.05). The worst values were observed when the ewes were supplemented with 15 and 30 g CG/kg DM. The N-efficiency was linearly increased (P < 0.01) with CG supplementation in drinking water.
Discussion
The main objective of supplementing with CG diluted in drinking water was to overcome the common peripartum decrease in DM intake by providing energy to support milk production during early lactation and throughout lactation. Polizel et al. (Reference Polizel, Susin, Gentil, Ferreira, de Souza, Freire, Pires A, Ferraz, Rodrigues and Eastridge2017) reported no effects of CG on DM intake and milk production in ewes (in late pregnancy and early lactation) fed up to 100 g CG/kg DM, a much greater dose compared to those from our study. However, our results suggest that CG supplementation in drinking water has a negative impact on DMI and, therefore, nutrient intake. Besides reducing DM intake, CG supplementation in drinking water also reduced absolute water intake. This result could suggest that crude glycerin has made the water less acceptable to ewes. However, no treatment effect was observed when the water intake was expressed as a percentage of body weight or metabolic body weight, a much better way to express such parameters. Another possible explanation is related to the metabolic water generated in gluconeogenesis. As a gluconeogenic compound, CG could have increased metabolic water production and reduced water intake in the ewes (Silva et al., Reference Silva, de Araujo, Santos, de Oliveira, Campos, Godoi, Gois, Perazzo, Ribeiro and Turco2021). Despite this effect, it is also worth mentioning that the water to DM intake ratio for all treatment values was within the reference range for ewes (NRC, 2007).
DMI reduction is undesirable because it usually affects milk production. We observed a linear decrease in milk production and milk component yield with CG supplementation. Osborne et al. (Reference Osborne, Odongo, Cant, Swanson and McBride2009) observed similar results when supplementing cows with CG (50 g kg DM−1) diluted in drinking water and also observed reduced DM intake. For transition dairy sheep, Sá et al. (Reference Sá, Borges, Macedo Junior, Santos, Cavalcanti, Alvarenga, Martins and Campolina2017) reported a quadratic effect of CG on DMI, where sheep supplemented with 15.0 g CG/kg showed higher greater DMI, and those supplemented with greater CG doses (30 and 45 g/kg) showed lower DMI.
These findings could be explained based on energy metabolism. Since animals feed primarily to meet their energy requirements (Mertens, Reference Mertens1997), if an animal is provided additional energy through another means it could decrease feed intake (Osborne et al., Reference Osborne, Odongo, Cant, Swanson and McBride2009). The reduction we observed in DMI coupled with lower milk production was not expected because glycerol (the main compound of CG) is thought to provide the mammary gland with substrates for milk synthesis. Glycerol has been previously reported to increase ruminal propionate production, a substrate for gluconeogenesis in the host animal that could provide glucose for mammary gland (Ferraro et al., Reference Ferraro, Mendoza, Miranda and Guti??rrez2009; Wang et al., Reference Wang, Liu, Huo, Yang, Dong, Huang and Guo2009; Abo El-Nor et al., Reference Abo El-Nor, AbuGhazaleh, Potu, Hastings and Khattab2010). Furthermore, glycerol can be absorbed directly through the rumen wall and used as a substrate for gluconeogenesis. However, our findings suggest that the additional energy provided by CG was insufficient to compensate for the reduction in DM intake. Thus, the mammary gland was probably not provided with sufficient substrate to support full milk secretion in ewes supplemented with CG in drinking water.
No difference among treatments was observed for blood glucose. In a previous study, DeFrain et al. (Reference DeFrain, Hippen, Kalscheur and Jardon2004) supplemented lactating dairy cows with 24.6 g glycerol/kg DM and observed a lower blood glucose concentration. This effect is not expected as glucose is the substrate for lactose synthesis. Lactose is the main osmotic and hence volume regulator, and we do not have a clear explanation. Thus, another factor other than glucose availability may have limited milk secretion in the ewes that we supplemented with CG.
That the N-efficiency was linearly increased with CG supplementation could be explained by the ruminal microbiota's putative enhancement in ammonia use. Indeed, Wang et al. (Reference Wang, Liu, Huo, Yang, Dong, Huang and Guo2009) have reported a lower ruminal N concentration in steers supplemented with glycerol. However, it is important to mention that microbial protein synthesis was not estimated in the present study.
In conclusion, our results suggest that dairy sheep can be supplemented with CG up to 15 g/kg DM in drinking water. Greater doses are not beneficial for feed intake, milk production, and the yield of milk components (protein, fat and lactose).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029923000377
Acknowledgments
The present study was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). F.S.S. was the recipient of a studentship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
