The effect of dietary pumpkin seed cake and extruded linseed on blood haemato-chemicals and milk quality in Alpine goats during early lactation

Introduction

In Slovenia, Austria, Hungary and Croatia, pumpkin seeds ( Cucurbita pepo L.) are used to produce edible oil and its by-products can be used in ruminant feeding. The nutritional value of pumpkin seed is based on high energy potential, due to high percentage of oil (40 %-60 %; Bavec et al., 2007) and high crude protein content (30.8 %; Kim et al., 2012). Pumpkin seed cake is produced by pressing of hull-less seeds with screw press. This method of production preserves bioactive components, such as vitamins, provitamins, phytosterols, phospholipids and squalene, which are, together with some fatty acids, very important nutritional factors of pumpkin seed cake (Rabrenović et al., 2014). Due to the high content of digestible crude protein compared to soybean meal (496.9 vs. 396.0 g/kg) and high concentration of unsaturated fatty acids of which oleic (C18:1 n-9, OA) and linoleic (C18:2 n-6, LA) are predominant, pumpkin seed cake is a good quality feedstuff for goats (Zdunczyk et al., 1999). In livestock diets soybean meal was more frequently replaced by alternative sources of plant protein (Gugołek et al., 2017), due to its doubtful origin and quality (Hewlett and Azeez, 2008). Antunović et al. (2018) found that pumpkin seed cake can be used in lambs' feeding as a partial replacement for soybean meal in terms of blood parameters and carcass traits. Besides, the inclusion of pumpkin seed cake in ruminants´ diets has not been extensively researched yet.

On contrary, numerous studies reported a high nutritive value of extruded linseed added in concentrates mixtures for dairy goats regarding n-3 fatty acids in milk, especially α-linolenic acid (C18:3 n-3, ALA) (Klir et al., 2012; Nudda et al., 2013; Bernard et al., 2015; Klir et al., 2017). Production of milk that is low in medium-chain saturated fatty acids and high in odd and branched-chain fatty acids, C18:1 c9, and C18:2 c9t11 (CLA, conjugated linoleic fatty acid) and PUFA could have benefits on human health (Castro et al., 2019). Although, an excessive fat from linseed may have deleterious effects on digestive efficiency, milk fat and protein content (Doreau et al., 2015), while unsaturated fatty acids (UFA) in diets may lead to oxidative stress (Wang et al., 2010). One of the most important determinants of goats´ production efficiency is monitoring of herds' health status which is of fundamental importance for disease detections that impair goats' production (Rumosa-Gwaze et al., 2012). The beginning of lactation is the most demanding period in terms of energy balance status and also health management of herd (Walsh et al., 2011). High-yielding goats require high amounts of energy during early lactation leading animals to mobilize energy from their body reserves (Teh et al., 1994).

Therefore, the objective of the present study was to research if pumpkin seed cake in diets of early lactating dairy goats influences haematological and biochemical parameters in blood and milk quality as compared with diets containing soybean meal and diet where extruded linseed partially replaced soybean meal.

Materials and methods

Bioethics Committee for Research on Animals of Faculty of Agrobiotechnical Sciences Osijek established that the present research is carried out under the legal provisions according to Animal Protection Act of Croatia (NN 133/06, NN 37/13 and NN 125/13). Animal handling practices meets the European Union Directive 2010/63/UE about the protection of animals used for the experiment.

Experimental design, animals, and feeding

The research was conducted with 28 French Alpine dairy goats at family farm in Osijek-Baranya County (Croatia). All goats had kidded within a period of one week. After 7 days of adaptation period to experimental diets, animals were set to experiment (12 ^th ± 3 days of lactation). Duration of the current experiment was 20 days, during early lactation, which was a part of larger experiment lasting 75 days (Klir et al., 2017). The sampling was conducted when goats were approximately in the 32 ^nd ± 3 days of lactation. All goats were healthy and in adequate body condition (score 2.87 ± 0.51) according to Russel (1991) when the experiment started, and had an average age of 3.6 ± 1.73 years. Goats were machine milked twice per day, in the morning and the evening, and kept together in the barn. All goats were fed ad libitum with grass and red clover hay (60 % DM Phleum pratense and Lolium multiflorium, and 40 % DM Trifolium pratense). In addition, each goat was supplemented with 1 kg/day of a concentrate mixture individually. Goats were fed in separated feeding troughs during milking, and had free access to fresh drinking water, while no refusals of concentrate were observed. Dietary treatments differed in the main source of crude protein and crude fat in the concentrate mixtures. Control group contained soybean meal (n = 9), while in ELS group 38.3 % of soybean was replaced with ELS (n = 10), and in PSC group soybean meal was completely replaced with PSC (n = 9; Table 1). The diet had an approximate hay to concentrate ratio of 70:30 (on DM basis).

Table 1. Ingredient composition of the concentrate mixtures used in diets for early lactating goats

ELS - concentrate mixture with partial replacement of soybean meal with extruded linseed, PSC - concentrate mixture with complete replacement of soybean meal with pumpkin seed cake

*Mineral-vitamin premix: Fe (iron sulphate monohydrate) 4000 mg, Cu (copper sulphate pentahydrate) 800 mg, Mn (manganese oxide) 3500 mg, Zn (zinc sulphate monohydrate) 5000 mg, I (potassium iodide) 80 mg, Co (cobalt sulphate heptahydrate) 20 mg, Se (sodium selenite) 15 mg, Mg (magnesium oxide) 5000mg; vitamin A 1 000 000 IU, vitamin D3 150 000 IU, vitamin E (α-tocopherol) 1500 mg, vitamin K3 (menadione sodium bisulfite) 50 mg, vitamin B1 (thiamine mononitrate) 100 mg, vitamin B2 200 mg, vitamin B6 200 mg, vitamin B12 1 mg, niacin 1000 mg, Ca-pantothenate 500 mg, choline chloride 10 000 mg

Analysis of feedstuffs

Composition of feed was determined with standard methods (AOAC, 2006). The crude protein concentrations were estimated from nitrogen content according to the Kjeldahl method with the steam distillation unit for Kjeldahl nitrogen (Behr, Germany). The crude fat concentrations were analysed using the Universal Extractions System B-811 (Buchi, Switzerland) . Metabolizable energy (ME, MJ/kg DM) of feed samples were calculated from gas production after 24 h of in vitro incubation with Hohenheim gas test according to Menke and Steingass (1987). All plant samples were digested with 10 mL of a 5:1 mixture of HNO ₃ and H ₂O ₂ at 180 °C for 60 min in microwave oven (CEM Mars 6, USA). The concentrations of mineral elements (Ca, P, Mg and Fe) in solutions of digested plant samples were determined by inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7500a, Agilent Technologies Inc., California, USA). The value for N-free extract (NFE) was calculated as: NFE (g/kg DM) = 1000 - (crude protein + crude lipid + crude fibre + ash). Fatty acids in dietary ingredients were determined by gas chromatography following the methodology of State Office of Agricultural Chemistry in Baden Württemberg (LaChemie P23-5-008, V. 01).

Table 2. Chemical composition and major fatty acid proportions of hay and concentrate mixtures used in the diets for early lactating goats

ELS - concentrate mixture with partial replacement of soybean meal with extruded linseed, PSC - concentrate mixture with complete replacement of soybean meal with pumpkin seed cake, DM - dry matter, NFE - nitrogen free extract, ME - metabolizable energy

Results and discussion

Milk yield and chemical composition of goat milk were not affected by the feeding treatment (Table 3). Similarly, the soybean replacement by PSC or partially by ELS did not result in any significant differences regarding the SCC and CFU. This indicated that PSC can be used as an alternative to soybean for the conditions of this study. Both PSC and ELS have a different fatty acids profile compared with soybean, but those changes in the diet did not affect either milk production. However, extruded linseeds addition in goats' diets lowered the milk yield, and increased the milk fat content in the study conducted by Chilliard et al. (2013).

The milk fatty acid profile has been changed when feeding goats with ELS and there were minor changes in PSC compared to Control (Table 4). The ELS and PSC diets did not influence the proportions of C6:0, C8:0 and C10:0, fatty acids responsible for the particular sensory properties of goats´ milk (Bessa et al., 2016) and health benefits providing better digestibility of goat milk (Zenebe et al., 2014). Bernard et al. (2015) found that SCFA, specifically C6:0, C8:0 and C10:0, were lowered in milk when feeding goats with 216 g/kg DM of extruded linseed in goats´ diets compared to control diets. The LCFA inhibit lipogenic enzymes of the mammary gland, leading to lowering of de novo synthesized fatty acid proportions (Chilliard et al., 2007). In the present study the proportion of ELS in the concentrate mixtures was probably not high enough to influence a lower proportion of these SCFA. Contrary, proportion of branched-chain fatty acids (BCFA) like C14:0 iso, and C15:0 iso, were significantly higher (P<0.05) when feeding goats with ELS compared to Control, while in PSC remained intermediate and unaffected. Supplementation of ruminants´ diet with different lipid source has relevant effect on ruminal bacteria community in goats (Cremonesi et al., 2018). Abuelfatah et al. (2016) reported that inclusion of linseed in the diet of goats at level of 10 or 20 g/100 g DM increases the molar proportion of acetate in rumen, which may be the reason for the increase of above stated milk fatty acids. Besides, Goetsch (2019) explained that numerous factors are responsible for microbial population in the rumen of dairy goats, including greater feed intake relative to body weight and shorter time of digesta residence in the rumen, and some other effects before treatment.

Table 3. Milk yield and chemical composition and fatty acid proportions in milk of early lactating goats fed with diets containing extruded linseed (ELS) and pumpkin seed cake (PSC)

SEM - standard error of mean; SCC - somatic cells count, CFU - number of bacterial formed colonies; ^a,bRow means with different superscripts differ significantly at P<0.05

Table 4. Fatty acid proportions in milk of early lactating goats fed with diets containing extruded linseed (ELS) and pumpkin seed cake (PSC)

¹Contains C18:1 t7 + t8 as a minor components, ²Contains C18:1 t10 as a minor component, ³Contains C18:1 t12 + t13 + t14 as a minor components, ⁴Contains C18:1 c14 as a minor component, ⁵Contains C19:0 a minor component, ⁶Contains C18:2 t9 t12 + t8 c12 + c9 t13 a minor components; SEM-standard error of mean; ^a,bRow means with different superscripts differ significantly at P < 0.05

Proportions of stearic (C18:0), OA, vaccenic (VA, C18:1 t11) and CLA remained unaffected by dietary treatments. Bernard et al. (2015) determined that addition of extruded linseed in combination with fish oil may be efficient in increasing VA and CLA proportions in milk. Although proportions of CLA, which is of the interest with respect to human health, were appropriate as compared to another studies, the amount of CLA consumed to improve human health still needs to be established (Hur et al., 2017).

In the present study ELS was abundant in ALA, which resulted in higher proportions of biohydrogenation metabolites, like C18:1 t15, C18:1 c15 and C18:2 t11 c15 compared to Control and PSC. Shingfield et al. (2010) reported that within putative pathway of ALA biohydrogenation C18:1 t15 and C18:1 c15 are produced as minor fatty acid metabolites, while in major ALA biohydrogenation pathway C18:2 t11 c15 is formed. Similar was reported in milk of ELS compared to Control and PSC of the present study.

Diets with PSC and ELS lowered LA compared to the Control, as a consequence of the higher LA concentration in the Control feed mixture containing soybean meal and extruded soybean (Table 1). This was reflected in lowered total n-6 fatty acids in ELS and PSC (Table 5). Proportion of ALA was the highest in ELS which resulted in higher ALA and total n-3 proportions, and lower LA/ALA ratio in milk of ELS goats compared to those of Control and PSC. The relationship between ALA intake and ALA proportion in milk is linear and transferred from diet to milk is close to 4.5 % (Doreau et al., 2015). According to the Commission Regulation EU (2010), a claim that food is a source of omega-3 fatty acids, may only be made when the product contains at least 0.3 g of ALA per 100 g, while food is high in omega-3 fatty acids when the product contains at least 0.6 g ALA per 100 g. As a consequence of greater ALA supply from the ELS diet proportion of EPA was higher compared to Control or PSC, as ALA is a precursor of EPA during the elongation.

Table 5. Groups of fatty acids in milk from early lactating goats fed with diets containing extruded linseed (ELS) and pumpkin seed cake (PSC)

*SCFA - short-chain fatty acids, MCFA - medium-chain fatty acids, LCFA - long-chain fatty acids, SFA - saturated fatty acids, BCFA - branched-chain fatty acids, OCFA - fatty acids with odd number of carbon atoms, MUFA - monounsaturated fatty acids, UFA - unsaturated fatty acids, PUFA - polyunsaturated fatty acids, LA - linoleic acid, ALA-α - linolenic acid; SEM - standard error of mean; ^a,bRow means with different superscripts differ significantly at P < 0.05

The concentration of globulins was the highest when PSC was fed and the lowest for the Control, while the rest of haemato-chemical parameters in goats’ blood resulted unchanged by the diets (Table 6). This observation could be due to high phenolic compounds in pumpkin seed cake of PSC diet as reported by Peričin et al. (2009). Polyphenol rich fraction from food have been shown to result in a significant increase in the IgM- and IgG-plaque forming cell responses of blood leukocytes (Hikosaka et al., 2007). The addition of feedstuff rich in crude fat in ruminants´ feeding may affect the production traits and health of ruminants (Nudda et al., 2013). All values of haemato-chemical parameters were within values determined in lactating Alpine goats by Antunović et al. (2013 and 2017).

Table 6. Haemato-chemical parameters from early lactating goats fed with diets containing extruded linseed (ELS) and pumpkin seed cake (PSC)

*RBC - erythrocytes, WBC - number of leukocytes, HGB - contents of haemoglobin, HCT - haematocrit, MCH - average haemoglobin content in erythrocytes, MCV - mean corpuscular volume, MCHC - mean haemoglobin concentration in erythrocytes, HDL - high-density lipoprotein, LDL - low-density lipoprotein, TGC - triglycerides, NEFA - non-esterified fatty acids, BHB - β-hydroxybutyrate, GPx - glutathione peroxidase, SOD - superoxide dismutase, AST - aspartate aminotransferase, ALT - alanine aminotransferase, GGT - gamma-glutamyl transferase, SEM - standard error of mean; ^a,bRow means with different superscripts differ significantly at P<0.05

It is well known that ruminants are susceptible to oxidative stress during pregnancy and lactation (Bernabucci et al., 2005), resulting in physiological state of metabolic stress that may cause diseases by changing the metabolic status (Antunović et al., 2013). The concentration of BHB in blood (≥0.8 mmol/L) and changes of the body score index (≤2.0) are specific indicators for assessment and prevention of metabolic disorders of highly productive lactating goats (Binev et al., 2014). Jahani-Moghadam et al. (2015) did not determine significant differences in the glucose, NEFA, BHB and urea concentrations in blood of cows fed ELS. Likewise, the results of the current study indicate the positive energy balance in early lactating dairy goats fed with ELS or PSC.

The ELS or PSC did not influence the activity of enzymes of antioxidant status. Bernabucci et al. (2005) reported that oxidative status is associated with energy status, which means that higher concentrations of NEFA and BHB are associated with lower activity of antioxidative enzymes in plasma. Additionally, Wang et al. (2010) established lower activity of SOD in plasma of cows fed UFA rich diets compared to cows fed with diets added SFA, while GPx activity and BHB concentration did not differ between groups. Owing to similar concentration of UFA in diets (Table 2) and stable energy status (Table 6), differences in the activity of the SOD and GPx enzyme were missing, and the antioxidant status was preserved.

Negative correlation was determined between the NEFA and the total SCFA or certain SCFA (Table 7). Short chain fatty acids which are synthesized de novo in the mammary gland decrease when plasma NEFA concentration increases (Craninx et al., 2008). The variability of milk C18:0 and OA concentration is linked to changes in the energy balance during the first 4 months of goats' lactation, since ruminant adipose tissue is very rich in palmitic, stearic, and OA (Chilliard et al., 2003). Established correlations result from the negative effect of LCFA mobilized from adipose tissue on de novo synthesized SCFA (Barber et al., 1997), as confirmed by present study on goats. Möller et al. (2018) determined the relationship between MCFA in hair and fat metabolism of cows. Dórea et al. (2017) found high sensitivities of milk C6:0, C8:0, C10:0 and C12:0 related to NEFA concentrations in cows' plasma. Accordingly, these authors determined threshold values to detect high NEFA concentrations in plasma, but when the proportion of these fatty acids are above these thresholds, the cow is classified as low concentrations of NEFA. In the present research a negative correlation was determined between the serum NEFA and C10:0 as well as C15:0 in goat milk while the other individual SCFA did not show any significant differences. Dórea et al. (2017) established that plasma NEFA concentrations may be predicted by linear regression models using rations C18:1 to C15:0 and C17:0 to C15:0 in cows´ milk. In the present study Pearson correlation showed positive correlation between both ratios in goat milk and serum NEFA, and only C17:0 to C15:0 ratio and BHB in serum. These correlations indicate low mobilization of lipids from adipose tissue in early lactating dairy goats in the current research.

Table 7. Correlations between the selected milk fatty acids and parameters of energy status in the serum of early lactating goats

NEFA - non-esterified fatty acids, BHB-beta-hydroxybutyrate, SCFA - short-chain fatty acids, MCFA - medium-chain fatty acids, LCFA - long-chain fatty acids

Conclusion

The current findings indicate that the replacement of soybean meal with pumpkin seed cake (PSC) or partially with extruded linseed (ELS) is feasible with respect to haemato-chemical parameters of the early lactating dairy goats. Moreover, these indicate adequate health, preserved antioxidant and nutritional status when goats were fed with PSC or ELS. Partially substituting soybean meal with ELS enhanced goat milk with ALA and total n-3 fatty acid proportions while compared to conventional diets, soybean meal replaced by PSC lowered linoleic acid in milk. In further study it is necessary to determine antioxidant status of milk which will contribute to better understanding whether PSC is suitable for goats´ feeding regarding milk quality.

Acknowledgements

The study was carried out within the research team Innovative breeding and technological processes in animal production (No. 1126) at Faculty of Agrobiotechnical Sciences Osijek.

Utjecaj pogače sjemenki bundeve i ekstrudiranog lana u obrocima na hematološko-biokemijske pokazatelje u krvi i na kvalitetu mlijeka alpske koze tijekom rane laktacije

References

https://doi.org/10.1016/j.aninu.2016.10.004

https://doi.org/10.5194/aab-60-409-2017

http://dx.doi.org/10.4314/sajas.v48i4.11

https://doi.org/10.24099/vet.arhiv.0780

https://doi.org/10.3168/jds.S0022-0302(05)72878-2

https://doi.org/10.1017/S1751731114003048

https://doi.org/10.1590/1678-457X.05315

https://doi.org/10.3390/ani9050205

https://doi.org/10.3168/jds.S0022-0302(03)73761-8

https://doi.org/10.1016/j.anifeedsci.2013.08.002

https://doi.org/10.3168/jds.2007-0656

https://doi.org/10.1017/S1751731117003433

Croatian Standards Institute (2007): HRN ISO 13366-2/Ispr.1:2007 (ISO 13366-2:2006)

Milk-Enumeration of somatic cells-Part 2: Guidance on the operation of fluoro-opto-electronic counters, Croatian Standards Institute, Zagreb, Croatia

Dórea, J.R.R., French, E.A.,Armentano, L.E. (2017): Use of milk fatty acids to estimate plasma nonesterified fatty acid concentrations as an indicator of animal energy balance. Journal of Dairy Science 100, 6164-6176.

https://doi.org/10.3168/jds.2016-12466

https://doi.org/10.1051/ocl/2015042

https://doi.org/10.1080/09712119.2019.1580585

https://doi.org/10.1080/1745039X.2017.1322796

https://doi.org/10.1002/ptr.2033

https://doi.org/10.1016/j.livsci.2016.11.016

https://doi.org/10.3168/jds.2014-8789

https://doi.org/10.4162/nrp.2012.6.1.21

https://doi.org/10.1017/S175173111700060X

Menke, K.H., Steingass, H. (1987): Schätzung des energetischen Futterwerts aus der in vitro mit Pansensaft bestimmten Gasbildung und der chemischen Analyse. II. Regressionsgleichungen. Übers Tierernährung 15, 59-94.

https://doi.org/10.1017/S0022029918000791

https://doi.org/10.1017/S1751731113000931

https://doi.org/10.1016/j.foodchem.2008.07.079

https://doi.org/10.1016/j.lwt.2013.10.019

https://doi.org/10.3168/jds.S0022-0302(94)76948-4

https://doi.org/10.3168/jds.2010-3054

https://doi.org/10.5829/idosi.ajn.2014.3.3.93210