Advertisement
Research| Volume 106, ISSUE 3, P1803-1814, March 2023

Replacing soybean meal with high-oil pumpkin seed cake in the diet of lactating Holstein dairy cows modulated rumen bacteria and milk fatty acid profile

Open AccessPublished:January 27, 2023DOI:https://doi.org/10.3168/jds.2022-22503

      ABSTRACT

      This research aimed to investigate the effects of replacing soybean meal with high-oil pumpkin seed cake (HOPSC) on ruminal fermentation, lactation performance, milk fatty acid, and ruminal bacterial community in Chinese dairy cows. Six multiparous Chinese Holstein cows at 105.50 ± 5.24 d in milk (mean ± standard deviation) and 36.63 ± 0.74 kg/d of milk yield were randomly allocated, in a 3 × 3 Latin square design, to 3 dietary treatments in which HOPSC replaced soybean meal. Group 1 was the basal diet with no HOPSC (0HOPSC); group 2 was a 50% replacement of soybean meal with HOPSC and dried distillers grains with solubles (DDGS; 50HOPSC), and group 3 was a 100% replacement of soybean meal with HOPSC and DDGS (100HOPSC). We found no difference in the quantity of milk produced or milk composition among the 3 treatment groups. Feed efficiency tended to increase linearly as more HOPSC was consumed. In addition, rumen fermentation was not influenced when soybean meal was replaced with HOPSC and DDGS; the relative abundance of ruminal bacteria at the phylum and genus levels was altered. We also observed that as the level of HOPSC supplementation increased, the relative abundance of Firmicutes and Tenericutes linearly increased, whereas that of Bacteroidetes decreased. However, with increasing HOPSC supplementation, the relative abundance of Ruminococcus decreased linearly at the genus level in the rumen, and the relative abundance of Prevotella showed a linear downward tendency. Changes in dietary composition and rumen bacteria had no significant effect on the fatty acid composition of milk. In conclusion, our results indicated that replacing soybean meal with a combination of HOPSC and DDGS can meet the nutritional needs of high-yielding dairy cows without adversely affecting milk yield and quality; however, the composition of rumen bacteria could be modified. Further study is required to investigate the effects of long-term feeding of HOPSC on rumen fermentation and performance of dairy cows.

      Key words

      INTRODUCTION

      The shortage of high-quality protein feed resources is a global problem (
      • Paula E.M.
      • Broderick G.A.
      • Danes M.A.C.
      • Lobos N.E.
      • Zanton G.I.
      • Faciola A.P.
      Effects of replacing soybean meal with canola meal or treated canola meal on ruminal digestion, omasal nutrient flow, and performance in lactating dairy cows.
      ;
      • Hao X.Y.
      • Yu S.C.
      • Mu C.T.
      • Wu X.D.
      • Zhang C.X.
      • Zhao J.X.
      • Zhang J.X.
      Replacing soybean meal with flax seed meal: Effects on nutrient digestibility, rumen microbial protein synthesis and growth performance in sheep.
      ;
      • Sezgin A.
      • Aydın B.
      Effect of replacing dietary soybean meal with pumpkin (Cucurbita pepo) seed cake on growth, feed utilization, haematological parameters and fatty acid composition of mirror carp (Cyprinus carpio).
      ), exacerbated by recent climate deterioration and other factors. Consequently, the price of dairy cow feedstuffs such as soybean meal, corn, and alfalfa hay continues to rise, especially in China, putting financial pressure on livestock producers. Therefore, attempts are being made to replace soybean meal with unconventional feed resources to reduce the cost per kilogram of milk (
      • Klir Z.
      • Castro-Montoya J.M.
      • Novoselec J.
      • Molkentin J.
      • Domacinovic M.
      • Mioc B.
      • Dickhoefer U.
      • Antunovic Z.
      Influence of pumpkin seed cake and extruded linseed on milk production and milk fatty acid profile in Alpine goats.
      ;
      • Paula E.M.
      • Broderick G.A.
      • Danes M.A.C.
      • Lobos N.E.
      • Zanton G.I.
      • Faciola A.P.
      Effects of replacing soybean meal with canola meal or treated canola meal on ruminal digestion, omasal nutrient flow, and performance in lactating dairy cows.
      ;
      • Hao X.Y.
      • Yu S.C.
      • Mu C.T.
      • Wu X.D.
      • Zhang C.X.
      • Zhao J.X.
      • Zhang J.X.
      Replacing soybean meal with flax seed meal: Effects on nutrient digestibility, rumen microbial protein synthesis and growth performance in sheep.
      ;
      • Jiang X.
      • Xu H.J.
      • Ma G.M.
      • Sun Y.K.
      • Li Y.
      • Zhang Y.G.
      Digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows fed a fermented corn gluten-wheat bran mixture as a substitute for soybean meal.
      ).
      Soybean meal is the most commonly used protein feed source for dairy cows on Chinese dairy farms due to its high protein content, desirable AA balance, and richness in limiting AA (
      • Gidlund H.
      • Hetta M.
      • Krizsan S.J.
      • Lemosquet S.
      • Huhtanen P.
      Effects of soybean meal or canola meal on milk production and methane emissions in lactating dairy cows fed grass silage-based diets.
      ;
      • Lopes J.C.
      • Harper M.T.
      • Giallongo F.
      • Oh J.
      • Smith L.
      • Ortega-Perez A.M.
      • Harper S.A.
      • Melgar A.
      • Kniffen D.M.
      • Fabin R.A.
      • Hristov A.N.
      Effect of high-oleic-acid soybeans on production performance, milk fatty acid composition, and enteric methane emission in dairy cows.
      ). To replace soybean meal, it is imperative that there are high quality requirements for the alternatives. Because China prohibits animal-derived protein for livestock feeding, plant-based feed ingredients with high protein content and adequate AA composition have been increasingly studied for ruminant nutrition. Among these feeds, canola meal (
      • Paula E.M.
      • Broderick G.A.
      • Danes M.A.C.
      • Lobos N.E.
      • Zanton G.I.
      • Faciola A.P.
      Effects of replacing soybean meal with canola meal or treated canola meal on ruminal digestion, omasal nutrient flow, and performance in lactating dairy cows.
      ), flaxseed meal (
      • Hao X.Y.
      • Yu S.C.
      • Mu C.T.
      • Wu X.D.
      • Zhang C.X.
      • Zhao J.X.
      • Zhang J.X.
      Replacing soybean meal with flax seed meal: Effects on nutrient digestibility, rumen microbial protein synthesis and growth performance in sheep.
      ), and pumpkin seed cake (
      • Klir Z.
      • Castro-Montoya J.M.
      • Novoselec J.
      • Molkentin J.
      • Domacinovic M.
      • Mioc B.
      • Dickhoefer U.
      • Antunovic Z.
      Influence of pumpkin seed cake and extruded linseed on milk production and milk fatty acid profile in Alpine goats.
      ) have received considerable attention in recent years. Pumpkins are in the Cucurbitaceae family, and “seed-used pumpkin” is the term for pumpkins whose seeds are the main part for processing. China has the world's largest planting area and highest pumpkin seed yield. In China, as in many European countries such as Slovenia, Austria, Hungary, and Croatia, large quantities of pumpkin seeds are processed into edible oil, and large quantities of byproducts are used as ruminant feed (
      • Šalavardić Ž.K.
      • Novoselec J.
      • Castro-Montoya J.M.
      • Šperanda M.
      • Đidara M.
      • Molkentin J.
      • Mioč B.
      • Dickhoefer U.
      • Antunović Z.
      The effect of dietary pumpkin seed cake and extruded linseed on blood haemato-chemicals and milk quality in Alpine goats during early lactation.
      ). The pumpkin seeds are usually cold-pressed to retain the active ingredients in pumpkin seed oil, including vitamins, provitamins, phytosterols, phospholipids, and squalene (
      • Sezgin A.
      • Aydın B.
      Effect of replacing dietary soybean meal with pumpkin (Cucurbita pepo) seed cake on growth, feed utilization, haematological parameters and fatty acid composition of mirror carp (Cyprinus carpio).
      ;
      • Li Y.
      • Wu Q.H.
      • Lv J.Y.
      • Jia X.M.
      • Gao J.X.
      • Zhang Y.G.
      • Wang L.
      Associations of protein molecular structures with their nutrient supply and biodegradation characteristics in different byproducts of seed-used pumpkin.
      ). This processing method makes pumpkin seed cake high in CP (39.5%) and fat (12.59%) and rich in highly unsaturated fatty acids (
      • Bardaa S.
      • Ben Halima N.
      • Aloui F.
      • Ben Mansour R.
      • Jabeur H.
      • Bouaziz M.
      • Sahnoun Z.
      Oil from pumpkin (Cucurbita pepo L.) seeds: Evaluation of its functional properties on wound healing in rats.
      ;
      • Sezgin A.
      • Aydın B.
      Effect of replacing dietary soybean meal with pumpkin (Cucurbita pepo) seed cake on growth, feed utilization, haematological parameters and fatty acid composition of mirror carp (Cyprinus carpio).
      ). Generally, dairy cows with high milk production receive 5 to 6% fat in their diets (
      • Bionaz M.
      • Vargas-Bello-Pérez E.
      • Busato S.
      Advances in fatty acids nutrition in dairy cows: From gut to cells and effects on performance.
      ). Supplementation of fat in ruminant diets not only increases dietary energy concentration but also has the potential to modulate the fatty acid profile of animal products, because the fat contained in the diet influences fatty acid proportions, hydrogenation level, and rumen microbial activity (
      • Mele M.
      • Buccioni A.
      • Serra A.
      • Antongiovanni M.
      • Secchiari P.
      Lipids of goat’s milk: Origin, composition and main sources of variation.
      ). Studies have shown that overconsumption of SFA and trans fats in the diet and insufficient intake of PUFA could increase the risk of cardiovascular disease in consumers of animal products (
      • Mozaffarian D.
      • Katan M.B.
      • Ascherio A.
      • Stampfer M.J.
      • Willett W.C.
      Trans fatty acids and cardiovascular disease.
      ;
      • Siri-Tarino P.W.
      • Sun Q.
      • Hu F.B.
      • Krauss R.M.
      Saturated fat, carbohydrate, and cardiovascular disease.
      ).
      Dietary changes can shift rumen microbial composition, structure, and fermentation (
      • Loor J.J.
      • Elolimy A.A.
      • Mccann J.C.
      Dietary impacts on rumen microbiota in beef and dairy production.
      ;
      • Wolff S.M.
      • Ellison M.J.
      • Hao Y.
      • Cockrum R.R.
      • Austin K.J.
      • Baraboo M.
      • Burch K.
      • Lee H.J.
      • Maurer T.
      • Patil R.D.
      • Ravelo A.
      • Taxis T.M.
      • Truong H.
      • Lamberson W.R.
      • Cammack K.M.
      • Conant G.C.
      Diet shifts provoke complex and variable changes in the metabolic networks of the ruminal microbiome.
      ). It was noted that dietary vegetable oil intake affects cellulolytic bacteria such as Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, and Butyrivibrio fibrisolvens, which are important in the biohydrogenation of PUFA (
      • Vargas-Bello-Pérez E.
      • Cancino-Padilla N.
      • Romero J.
      • Garnsworthy P.C.
      Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils.
      ). And, like UFA, SFA such as stearic acid and palmitic acid are also somewhat toxic to Prevotella ruminicola (
      • Enjalbert F.
      • Combes S.
      • Zened A.
      • Meynadier A.
      Rumen microbiota and dietary fat: A mutual shaping.
      ). Compared with soybean meal, the UFA in pumpkin seed cake are mainly oleic acid and linoleic acid, and the content is high (
      • Zdunczyk Z.
      • Minakowski D.
      • Frejnagel S.
      • Flis M.
      Comparative study of the chemical composition and nutritional value of pumpkin seed cake, soybean meal and casein.
      ). Therefore, the aim of this study was to evaluate whether replacing soybean meal with pumpkin seed cake would affect the milk fatty acid profile and explore the effect of UFA on rumen fermentation and microflora when pumpkin seed cake is largely substituted for soybean meal in high-yielding dairy cow formulations. In high-yielding dairy cows' diets, substantial replacement of soybean meal with high-oil pumpkin seed cake (HOPSC) results in an increased dietary UFA content that may affect the rumen microflora when the diet contains 5 to 6% fat. An in-depth understanding of the relationship between the structure and function of the rumen microflora under different dietary conditions can predict the rumen health of dairy cows and provide interventions for improving dairy cow production performance (
      • Cammack K.M.
      • Austin K.J.
      • Lamberson W.R.
      • Conant G.C.
      • Cunningham H.C.
      Ruminant Nutrition Symposium: Tiny but mighty: The role of the rumen microbes in livestock production.
      ). We previously conducted a systematic study of different byproducts of seed-used pumpkin and found that replacing soybean meal with a small amount of pumpkin seed cake did not affect the performance of dairy cows (
      • Li Y.
      • Zhang G.N.
      • Fang X.P.
      • Zhao C.
      • Wu H.Y.
      • Lan Y.X.
      • Che L.
      • Sun Y.K.
      • Lv J.Y.
      • Zhang Y.G.
      • Pan C.F.
      Effects of replacing soybean meal with pumpkin seed cake and dried distillers grains with solubles on milk performance and antioxidant functions in dairy cows.
      ). However, the effects of feeding large amounts of pumpkin seed cake as a replacement for soybean meal in high-yielding dairy cow formulations on lactating performance, rumen fermentation, and rumen microflora have not been reported. Based on the above theories and studies, we hypothesized that in a high-yielding dairy cow diet, partial or total replacement of soybean meal with HOPSC would not negatively affect production performance and rumen fermentation, and would improve the content and composition of fatty acids in milk.
      Therefore, this feeding experiment aimed to investigate the effects of replacing soybean meal with HOPSC on milk production, rumen fermentation, rumen bacteria, and milk fatty acid profile in dairy cows.

      MATERIALS AND METHODS

      Preparation of HOPSC

      The HOPSC (ether extract = 14.16%, DM basis) was prepared at the Ruminant Nutrition Laboratory of Northeast Agricultural University (Harbin, China). The 3 most commonly grown pumpkin seeds (Yinhui No. 1, Yinhui No. 2, and Jinhui No. 1) were collected from Heilongjiang Hexing Agricultural Products Company Ltd. The pumpkin seeds were processed using a physical cold-pressing method and a spiral oil extractor (KYL-380 oil extractor) with temperature controlled at 70°C, 7.5% moisture content, rotation rate of 36 revolutions/min, and 47% oil extraction. The HOPSC was then obtained after oil extraction of the pumpkin seeds.

      Animals, Experimental Design, and Diets

      This study was approved and conducted in accordance with the regulations of the Animal Care Advisory Committee of Northeast Agricultural University (NEAU-[2011]-9).
      In a 21-d trial, 6 multiparous (parity = 3) Chinese Holstein dairy cows were randomly assigned to a replicated 3 × 3 Latin square design. Cows were allowed to adapt to the diet within the first 16 d, and data and sample collection was carried out for 5 d. The animals were distributed in squares to balance for differences in DIM, milk production, and parity, resulting in 2 squares (mean ± SD; square 1: 682 ± 7.8 kg of BW, 106 ± 4.2 DIM, 36.2 ± 0.81 kg of milk/d; square 2: 680 ± 8.0 kg of BW, 105 ± 7.0 DIM, 37.0 ± 0.47 kg of milk/d). Experimental diets (Table 1) were isocaloric and isonitrogenous and formulated at a 48:52 forage:concentrate ratio on a DM basis according to the Cornell-Penn-Miner dairy model (version 3.0.10; Cornell University, Ithaca, NY; University of Pennsylvania, Kennett Square, PA; and William H. Miner Agricultural Research Institute, Chazy, NY) to meet the animals' nutrient requirements.
      Table 1Formulation of dietary treatments and their chemical composition (% of DM unless otherwise stated)
      ItemTreatment
      0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      0HOPSC50HOPSC100HOPSC
      Ingredient
       Alfalfa hay18.6718.6718.67
       Corn silage29.7829.7829.78
       Ground corn27.1027.1027.10
       Cottonseed meal3.563.563.56
       Soybean meal
      Soybean meal contained (on a DM basis): 88.1% DM, 43.4% CP, 12.5% NDF, 8.23% ADF, 1.88% ether extract, 2.62% Lys, 0.59% Met; high-oil pumpkin seed cake contained (on a DM basis): 87.3% DM, 53.2% CP, 21.4% NDF, 8.11% ADF, 14.16% ether extract, 2.70% Lys, 1.28% Met; DDGS contained (on a DM basis): 88.7% DM, 27.6% CP, 28.4% NDF, 23.5% ADF, 3.11% ether extract, 0.76% Lys, 0.67% Met. Megalac, rumen-protected fat supplement (Volac Wilmar Feed Ingredients Ltd.).
      17.788.890.00
       High-oil pumpkin seed cake
      Soybean meal contained (on a DM basis): 88.1% DM, 43.4% CP, 12.5% NDF, 8.23% ADF, 1.88% ether extract, 2.62% Lys, 0.59% Met; high-oil pumpkin seed cake contained (on a DM basis): 87.3% DM, 53.2% CP, 21.4% NDF, 8.11% ADF, 14.16% ether extract, 2.70% Lys, 1.28% Met; DDGS contained (on a DM basis): 88.7% DM, 27.6% CP, 28.4% NDF, 23.5% ADF, 3.11% ether extract, 0.76% Lys, 0.67% Met. Megalac, rumen-protected fat supplement (Volac Wilmar Feed Ingredients Ltd.).
      0.005.5612.00
       Dried distillers grains with solubles (DDGS)
      Soybean meal contained (on a DM basis): 88.1% DM, 43.4% CP, 12.5% NDF, 8.23% ADF, 1.88% ether extract, 2.62% Lys, 0.59% Met; high-oil pumpkin seed cake contained (on a DM basis): 87.3% DM, 53.2% CP, 21.4% NDF, 8.11% ADF, 14.16% ether extract, 2.70% Lys, 1.28% Met; DDGS contained (on a DM basis): 88.7% DM, 27.6% CP, 28.4% NDF, 23.5% ADF, 3.11% ether extract, 0.76% Lys, 0.67% Met. Megalac, rumen-protected fat supplement (Volac Wilmar Feed Ingredients Ltd.).
      0.003.335.78
       Premix
      The premix contained (on a DM basis): 99.17% ash, 14.25% Ca, 5.40% P, 4.93% Mg, 0.05% K, 10.64% Na, 2.95% Cl, 0.37% S, 12 mg/kg Co, 500 mg/kg Cu, 500 mg/kg Fe, 25 mg/kg I, 800 mg/kg Mn, 10 mg/kg Se, 1 800 mg/kg Zn, 180,000 IU/kg vitamin A, 55,000 IU/kg vitamin D, and 1,500 IU/kg vitamin E.
      2.222.222.22
       Megalac
      Soybean meal contained (on a DM basis): 88.1% DM, 43.4% CP, 12.5% NDF, 8.23% ADF, 1.88% ether extract, 2.62% Lys, 0.59% Met; high-oil pumpkin seed cake contained (on a DM basis): 87.3% DM, 53.2% CP, 21.4% NDF, 8.11% ADF, 14.16% ether extract, 2.70% Lys, 1.28% Met; DDGS contained (on a DM basis): 88.7% DM, 27.6% CP, 28.4% NDF, 23.5% ADF, 3.11% ether extract, 0.76% Lys, 0.67% Met. Megalac, rumen-protected fat supplement (Volac Wilmar Feed Ingredients Ltd.).
      0.890.890.89
      Chemical composition
       CP17.0017.0017.16
       RDP (% of CP)62.8562.9863.11
       RUP (% of CP)37.1537.0236.89
       NDF27.7428.8529.91
       ADF17.0417.5918.01
       Starch27.0226.9326.75
       Ether extract3.434.175.00
       Forage NDF21.7421.7421.74
       Physically effective NDF22.3122.2922.33
       NEL
      NEL was estimated according to CPM Dairy 3.0.10 (Wei et al., 2018).
      (Mcal/kg of DM)
      1.711.711.72
       Lys (% of requirement)122116112
       Met (% of requirement)108113118
       ME for milk
      ME-allowable milk yield predictions from CPM Dairy (Tedeschi et al., 2008).
      (kg/d)
      41.842.142.4
       MP for milk
      MP-allowable milk yield predictions from CPM Dairy (Tedeschi et al., 2008).
      (kg/d)
      40.041.843.9
      Fatty acid (g/100 g of total fatty acid)
       C14:00.390.390.39
       C16:018.1619.3720.55
       C16:10.550530.52
       C17:00.170.170.16
       C18:02.582.662.75
       C18:1 cis-921.9722.4723.56
       C18:2 cis-9,cis-1242.6643.5944.67
       C18:38.668.658.62
       C20:00.510.500.49
       C20:10.230.230.22
       C22:00.440.440.45
       C22:20.460.450.45
      1 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      2 Soybean meal contained (on a DM basis): 88.1% DM, 43.4% CP, 12.5% NDF, 8.23% ADF, 1.88% ether extract, 2.62% Lys, 0.59% Met; high-oil pumpkin seed cake contained (on a DM basis): 87.3% DM, 53.2% CP, 21.4% NDF, 8.11% ADF, 14.16% ether extract, 2.70% Lys, 1.28% Met; DDGS contained (on a DM basis): 88.7% DM, 27.6% CP, 28.4% NDF, 23.5% ADF, 3.11% ether extract, 0.76% Lys, 0.67% Met. Megalac, rumen-protected fat supplement (Volac Wilmar Feed Ingredients Ltd.).
      3 The premix contained (on a DM basis): 99.17% ash, 14.25% Ca, 5.40% P, 4.93% Mg, 0.05% K, 10.64% Na, 2.95% Cl, 0.37% S, 12 mg/kg Co, 500 mg/kg Cu, 500 mg/kg Fe, 25 mg/kg I, 800 mg/kg Mn, 10 mg/kg Se, 1 800 mg/kg Zn, 180,000 IU/kg vitamin A, 55,000 IU/kg vitamin D, and 1,500 IU/kg vitamin E.
      4 NEL was estimated according to CPM Dairy 3.0.10 (
      • Wei Z.
      • Zhang B.
      • Liu J.
      Effects of the dietary nonfiber carbohydrate content on lactation performance, rumen fermentation, and nitrogen utilization in mid-lactation dairy cows receiving corn stover.
      ).
      5 ME-allowable milk yield predictions from CPM Dairy (
      • Tedeschi L.O.
      • Chalupa W.
      • Janczewski E.
      • Fox D.G.
      • Sniffen C.J.
      • Munson R.
      • Kononoff P.J.
      • Boston R.C.
      Evaluation and application of the CPM Dairy nutrition model.
      ).
      6 MP-allowable milk yield predictions from CPM Dairy (
      • Tedeschi L.O.
      • Chalupa W.
      • Janczewski E.
      • Fox D.G.
      • Sniffen C.J.
      • Munson R.
      • Kononoff P.J.
      • Boston R.C.
      Evaluation and application of the CPM Dairy nutrition model.
      ).
      Cows were fed (1) control diet, containing 17.78% soybean meal on a DM basis (0HOPSC), (2) 0HOPSC with half replacement of soybean meal with HOPSC (5.56% of DM) and dried distillers grains with solubles (DDGS, 3.33% of DM; 50HOPSC), or (3) 0HOPSC with full replacement of soybean meal with HOPSC (12.00% of DM) and DDGS (5.78% of DM; 100HOPSC). The cows were housed in tiestalls, had free access to water, were fed twice daily at 0630 and 1830 h at 5% refusals, and were milked twice daily at 0600 and 1800 h.

      Measurements and Chemical Analysis

      TMR Chemical Composition

      Samples of TMR were collected daily from 14 to 21 d. The samples were oven-dried at 55°C for 48 h, ground and sieved through a 1-mm mesh, and stored at −20°C to determine chemical compositions. The samples were analyzed using the wet chemical method, in which ether extract (method 920.39), CP (method 984.13), ash (method 942.05), and DM (method 930.15) content were determined according to the
      • AOAC International
      Official Methods of Analysis.
      . Contents of NDF and ADF were determined according to the methods of
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      using heat-stable α-amylase treatment. A total starch kit (product no: KTSTA; Megazyme International Ireland Ltd.) was used to measure the starch content of TMR, and the AA content in TMR was analyzed at the Heilongjiang Academy of Agricultural Sciences (Harbin, China) using a fully automated AA detector (S-433D; Secam Scientific Instrument Co. Ltd.). The specific steps are the same as those described by
      • Jiang X.
      • Xu H.J.
      • Ma G.M.
      • Sun Y.K.
      • Li Y.
      • Zhang Y.G.
      Digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows fed a fermented corn gluten-wheat bran mixture as a substitute for soybean meal.
      .

      Milk and TMR Fatty Acid Composition by FAME

      Cows were milked twice daily for 3 consecutive days from 17 to 19 d to estimate milk yield. Milk samples (50 mL) were stored at −20°C to determine the fatty acid composition, and another 50 mL of milk was stored at 4°C for protein, fat, lactose, and MUN concentrations, and SCC analysis. Milk samples were tested using a 4-channel spectrophotometer (MilkoScan; Foss Electric) at the Heilongjiang Academy of Agricultural Reclamation (Harbin, China). Further, the milk fatty acid content was determined using FAME. In this method, milk fat was first extracted, and the fatty acids were methylated with methanolic sodium methoxide in the presence of methyl acetate. The FAME were then determined by GC using a flame-ionization detector (GC-8A; Shimadzu Corp.) and an SP 2380 capillary column (60 m × 0.25 µm × 0.25 mm; Supelco;
      • Bayat A.R.
      • Vilkki J.
      • Razzaghi A.
      • Leskinen H.
      • Kettunen H.
      • Khurana R.
      • Brand T.
      • Ahvenjärvi S.
      Evaluating the effects of high-oil rapeseed cake or natural additives on methane emissions and performance of dairy cows.
      ). The fatty acid content of TMR was determined by the same procedure as used in milk. However, before GC analysis, the samples were air-dried by a single-extraction methylation procedure, and FAME were prepared using chloroform and methanol sulfuric acid (
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Ärölä A.
      • Nurmela K.V.V.
      • Huhtanen P.
      • Griinari J.M.
      Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows.
      ).

      Rumen Fermentation Characteristics

      Three hours after the morning feeding on d 20 and 21, rumen fluid was collected from the cows orally using a stomach tube. The first 150 to 200 mL of rumen fluid was discarded to prevent contamination with saliva. Immediately after sampling, rumen fluid (100–200 mL) was filtered through a 4-layer gauze. The filtrate (50 mL) was stored in a cryogenic vial at −20°C for subsequent analysis of ammonia-N and VFA. The concentration of ammonia-N was determined by the phenol-hypochlorite method (
      • Broderick G.A.
      • Kang J.H.
      Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media.
      ), and the concentration of VFA was determined by GC (GC-8A; Shimadzu Corp.;
      • Stewart C.S.
      • Duncan S.H.
      The effect of avoparcin on cellulolytic bacteria of the ovine rumen.
      ).

      Rumen Microbial DNA Extraction and 16S rRNA Gene Sequencing

      Rumen fluid (50 mL) was immediately frozen in liquid nitrogen and stored at −80°C in a cryogenic vial. DNA was extracted from the rumen fluid samples using MN NucleoSpin 96 Soil DNA kit (Gene Company Ltd.). The V3-V4 region of 16S RNA was amplified by the 2-step PCR amplification method on a Veriti 96-well PCR instrument (9902; Applied Biosystems Inc.). The primers used were 338F (5′-ACTCCTRCGGGAGGCAGCAGCAG-3′) and 806R (5′-GGACTACCVGGGTATCtaat-3′) (
      • Mao S.Y.
      • Zhang M.L.
      • Liu J.H.
      • Zhu W.Y.
      Characterising the bacterial microbiota across the gastrointestinal tracts of dairy cattle: Membership and potential function.
      ). The PCR products in the first step were then used as templates for Solexa PCR amplification in the second step, as described by
      • Jiang X.
      • Xu H.J.
      • Ma G.M.
      • Sun Y.K.
      • Li Y.
      • Zhang Y.G.
      Digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows fed a fermented corn gluten-wheat bran mixture as a substitute for soybean meal.
      , and DNA was purified with Omega DNA purification columns on 1.8% agarose gel. The quantification of purified PCR products was determined using Quant-iT PicoGreen dsDNA Assay Kit (Gene Company Ltd.), followed by sequencing of DNA fragments through double-terminal (paired-end) sequencing on an Illumina MiSeq platform (Wuhan Frasergen Bioinformatics Co. Ltd.). Data sequencing was performed using Quantitative Insights into Microbial Ecology (QIIME, v1.8.0 http://qiime.org/;
      • Caporaso J.G.
      • Kuczynski J.
      • Stombaugh J.
      • Bittinger K.
      • Bushman F.D.
      • Costello E.K.
      • Fierer N.
      • Peña A.
      • Goodrich J.K.
      • Gordon J.I.
      • Huttley G.A.
      • Kelley S.T.
      • Knights D.
      • Koenig J.E.
      • Ley R.E.
      • Lozupone C.A.
      • McDonald D.
      • Muegge B.D.
      • Pirrung M.
      • Reeder J.
      • Sevinsky J.R.
      • Turnbaugh P.J.
      • Walters W.A.
      • Widmann J.
      • Yatsunenko T.
      • Zaneveld J.
      • Knight R.
      QIIME allows analysis of high-throughput community sequencing data.
      ). Using Mothur software, the species abundance and diversity of perational taxonomic units were counted at the phylum and genus classification levels. The α-diversity index of rumen fluid bacteria was detected by QIIME software; 4 indexes (Chao1, ACE, Shannon, and Simpson) were detected and compared. The rumen microbiome was also compared as a β-diversity metric based on principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

      Calculations and Statistical Analysis

      All data were screened for normality using the UNIVARIATE procedure of SAS and analyzed in a 3 × 3 Latin square design using the Proc Mixed procedure of SAS (version 9.4; SAS Institute Inc.), according to the model
      Yijkm = μ + Si + C(S)ij + Pk + Tm + Eijkm,


      where Yijkm is the observation, μ is the overall mean, Si is the fixed effect of square i, C(S)ij is the random effect of cow j within square i, Pk is the fixed effect of period k, Tm is the fixed effect of treatment m, and Eijkm is the residual error. Orthogonal polynomial contrasts were also used to analyze the linear and quadratic effects of increasing HOPSC supplementation on each variable. Significance was declared at P ≤ 0.05 and considered a trend when 0.05 < P ≤ 0.10.

      RESULTS

      Diet Composition

      The diets in this experiment contained equal amounts of roughage but different proportions of soybean meal, HOPSC, and DDGS; also, diets were formulated to be isocaloric and isonitrogenous by adjusting the ratio of different feed ingredients. The results of this study indicated that as the proportion of HOPSC and DDGS in the formula increased, the fat, NDF, and ADF contents of the diets gradually increased. Metabolizable protein– and ME-allowable milk yield for 40-kg predictions from CPM Dairy were sufficient. In addition, there was no significant change in the fatty acid contents of the 3 diets. The proportions (g/100 g of fatty acids) of C16:0, C18:0, cis-9 C18:1 and cis-9,cis-12 C18:2 in the diet increased gradually as the proportion of HOPSC in the formula increased.

      Lactation Performance

      As shown in Table 2, the replacement of soybean meal with a combination of HOPSC and DDGS did not have any negative effects on lactation performance and milk composition of cows. Also, no significant differences were observed in DMI, milk production, ECM, FCM, or yields of milk fat, protein, and lactose among cows fed different diets (P > 0.10). The contents of fat, protein, lactose, MUN, and SCC in milk did not differ between the 50HOPSC, 100HOPSC, and 0HOPSC groups (P > 0.10; Table 2). However, feed efficiency (milk/DMI and ECM/DMI) of cows in the control group (0HOPSC) showed linear upward tendencies (P = 0.088 and P = 0.079, respectively).
      Table 2Milk production, milk composition, and feed efficiency of lactating dairy cows fed 3 diets
      ItemTreatment
      0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      SEMP-value
      0HOPSC50HOPSC100HOPSCLinearQuadratic
      DMI24.9724.6724.430.5890.110.90
      Production (kg/d)
       Milk36.5336.8336.930.6410.320.77
       ECM39.6239.6539.980.7800.510.75
       4% FCM35.1735.1335.370.6240.690.75
       Fat1.371.361.370.02690.920.61
       Protein1.241.261.270.03730.170.90
       Lactose1.831.861.860.05000.310.66
      Composition
       Fat (%)3.753.693.670.05030.590.39
       Protein (%)3.413.413.440.05130.330.64
       Lactose (%)5.015.045.040.06890.460.63
       MUN (mg/dL)13.8713.6714.000.2780.680.34
       SCC (×103 cells/mL)151.03153.30155.474.5990.400.99
      Feed efficiency
       Milk/DMI1.471.491.510.02400.0880.84
       ECM/DMI1.591.611.630.02350.0790.81
      1 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.

      Ruminal Fermentation

      Table 3 shows that feeding different experimental diets did not affect rumen fermentation patterns, ammonia nitrogen and total VFA contents, molar ratios of various VFA, or acetate-to-propionate ratio among the different dietary groups (P > 0.10).
      Table 3Rumen fermentation characteristics of lactating dairy cows fed 3 diets
      ItemTreatment
      0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      SEMP-value
      0HOPSC50HOPSC100HOPSCLinearQuadratic
      Ammonia-N (mg/dL)12.6312.5812.650.3520.970.89
      Total VFA (mmol/L)96.2195.8396.543.0470.850.73
      VFA profile (mol/100 mol)
       Acetate64.6565.0764.731.9400.880.38
       Propionate21.6821.4721.770.9050.910.71
       Isobutyrate1.031.051.040.07180.730.78
       Butyrate10.8710.6710.711.4230.840.86
       Isovalerate0.860.880.880.07900.760.73
       Valerate0.880.850.870.06110.890.69
       Acetate:propionate3.013.043.000.2030.920.75
      1 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.

      Bacterial Communities

      Using high-throughput sequencing of the rumen fluid DNA from the cows fed different diets, we observed that replacing soybean meal with varying ratios of HOPSC and DDGS combinations did not affect bacterial α-diversity indices (ACE, Chao1, Shannon, and Simpson, P > 0.10; Table 4). The relative abundances (>1%) of bacterial phyla and genera in ruminal fluid are shown in Table 5 and Figure 1. For the bacterial phylum-level community composition, we observed that the relative abundances of Firmicutes (P = 0.024) and Tenericutes (P = 0.042) increased linearly with HOPSC supplementation, and that of Bacteroidetes (P = 0.042) decreased linearly. In contrast, the relative abundance of Ruminococcus (P = 0.045) decreased linearly with HOPSC supplementation, and that of the Prevotella population showed a linear downward tendency (P = 0.076). Additionally, PCA and PLS-DA were used to analyze the bacterial community structure in rumen fluid of lactating dairy cows, and the results indicated that bacterial abundance differed among treatment groups (Figure 2).
      Table 4Comparison of bacterial α-diversity indices in rumen fluid of lactating dairy cows fed 3 diets
      IndexTreatment
      0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      SEMP-value
      0HOPSC50HOPSC100HOPSCLinearQuadratic
      ACE2001.232,357.422,241.73302.750.250.21
      Chao 11906.962,239.052,077.60274.270.460.27
      Shannon8.708.818.490.3610.560.49
      Simpson0.00920.00870.0130.002670.290.41
      1 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      Table 5Relative abundance (>1%) of bacterial phyla and genera in the rumen fluid of lactating dairy cows fed 3 diets
      ItemTreatment
      0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      SEMP-value
      0HOPSC50HOPSC100HOPSCLinearQuadratic
      Phylum
      Firmicutes41.5048.1049.132.3340.0240.21
      Bacteroidetes44.0036.1332.433.0060.0420.43
      Tenericutes3.175.236.230.8540.0010.025
      Cyanobacteria3.032.302.270.8170.300.57
      Actinobacteria1.771.733.930.9200.110.28
      Proteobacteria3.002.701.271.3500.400.74
      Spirochaetes1.631.873.000.7050.200.59
      Genus
      Prevotella20.7015.179.733.6150.0760.99
      Sharpea4.535.138.502.0470.230.56
      RFN205.103.704.471.9580.750.55
      Succiniclasticum2.304.101.371.2990.630.20
      Ruminococcus3.701.771.230.6300.0450.40
      Treponema1.301.602.800.6240.130.54
      Shuttleworthia1.172.201.730.8280.520.34
      Bifidobacterium1.330.801.870.2230.120.046
      Butyrivibrio1.201.071.470.1480.190.14
      Coprococcus1.031.600.930.3710.770.088
      Succinivibrio2.001.030.431.1850.390.90
      YRC221.030.601.600.3310.230.10
      Lachnospira0.431.730.800.4920.530.071
      Clostridium0.830.631.370.6140.480.47
      1 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      Figure thumbnail gr1
      Figure 1Relative abundance (%) of bacteria at the phylum (left) and genus (right) level in the rumen fluid of lactating dairy cows fed 3 diets: 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      Figure thumbnail gr2
      Figure 2Principal component (PC) analysis and partial least squares discriminant analysis (PLS-DA) of bacterial community structure in the rumen fluid of lactating dairy cows fed 3 diets: 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.

      Milk Fatty Acid Composition

      The proportions (g/100 g of fatty acids) of different classifications of fatty acids in the milk of dairy cows are listed in Table 6. A total of 23 fatty acids were identified under the 3 dietary conditions; among them, the highest contents in milk were of C14:0, C16:0, C18:0, and cis-9 C18:1. However, we observed no significant differences (P > 0.10) in the milk fatty acids among the 3 dietary groups.
      Table 6Fatty acid (FA) composition in milk of lactating dairy cows fed 3 diets
      Item (g/100 g of FA)Treatment
      0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.
      SEMP-value
      0HOPSC50HOPSC100HOPSCLinearQuadratic
      4:03.583.663.560.07990.760.11
      6:02.162.172.180.07970.680.95
      8:01.321.391.350.09850.720.41
      10:03.783.833.760.08120.810.39
      11:00.0810.0760.0780.003160.560.36
      12:04.214.334.270.07090.600.30
      13:00.0780.0730.0740.003920.450.56
      14:013.2713.3213.350.4890.740.94
      cis-9 14:10.0980.0930.0950.005710.740.61
      15:00.0760.0760.0800.002980.360.59
      cis-10 15:10.0260.0260.0300.002350.250.50
      16:029.9430.9930.181.0740.760.20
      cis-9 16:10.0310.0330.0350.003730.250.91
      17:00.0610.0580.0650.004140.430.23
      cis-10 17:10.210.210.220.01650.830.90
      18:0011.2711.3211.350.6870.880.97
      trans-9 18:10.0370.0380.0350.005890.300.17
      cis-9 18:116.2716.3216.350.7700.870.97
      cis-9,cis-12 18:21.361.341.370.1080.930.83
      cis-9,trans-11 18:20.430.400.500.04510.250.27
      trans-10,cis-12 18:20.0330.0340.0360.004120.430.84
      cis-9,cis-12,cis-15 18:30.360.360.360.01460.990.95
      cis-11 20:10.250.250.240.01620.700.88
      1 0HOPSC = basal diet; 50HOPSC = 50% replacement of soybean meal with high-oil pumpkin seed cake (HOPSC); 100HOPSC = 100% replacement of soybean meal with HOPSC.

      DISCUSSION

      Milk Performance and Rumen Fermentation Characteristics

      The premise for the application of unconventional feed resources in ruminant production is that it does not affect the health and lactation performance of dairy cows. Because of the complex ruminant digestive system, especially protein metabolism in the rumen, the dietary CP content and protein components were considered in our studies. Therefore, we ensured that the 3 experimental diets contained the same levels of nitrogen and energy and the same ratios of RDP and RUP (Table 1). As in our previous study, a combination of HOPSC and DDGS with high RUP content was used to replace soybean meal to ensure stability of the diet's nutrient content (
      • Li Y.
      • Zhang G.N.
      • Fang X.P.
      • Zhao C.
      • Wu H.Y.
      • Lan Y.X.
      • Che L.
      • Sun Y.K.
      • Lv J.Y.
      • Zhang Y.G.
      • Pan C.F.
      Effects of replacing soybean meal with pumpkin seed cake and dried distillers grains with solubles on milk performance and antioxidant functions in dairy cows.
      ), thereby ensuring that production performance of dairy cows in the different treatment groups was not affected. However, the amount of pumpkin seed cake included was greater than that used in previous studies. Surprisingly, the protein feed based on HOPSC and DDGS supported 40 kg of milk production, making our follow-up research more realistic and scientifically valuable.
      The content of ether extract in pumpkin seed cake may vary depending on the pumpkin seed oil content and the oil pressing process. The oil content of pumpkin seed cake in this study obtained by the cold pressing method is higher than that obtained by the hot pressing method and leaching method. An increase in fat content and various fatty acids in the diets was observed with the addition of HOPSC. Considering the effect of increasing fat and dietary UFA content on rumen fermentation in dairy cows, we controlled the fat content in the diet such that it did not exceed 5%. However, changes in dietary fatty acid content may still be the main cause of changes in the relative abundance of rumen bacteria (Table 5). However, probably because of the short feeding time, changes in rumen microorganisms were not reflected in changes in product performance or rumen fermentation parameters. In particular, changes in partitioning of fatty acids between adipose tissue and mammary glands were not evaluated during the short experimental period (21 d) of the current study.
      The production of VFA and ammonia-N in the rumen can cause changes in milk production and composition. Replacement of soybean meal with HOPSC and DDGS resulted in similar nutritional levels among the treatment groups, ensuring the same feed intake, metabolic protein production, and VFA production and composition in the rumen, as well as similar milk yield and milk composition. Some researchers have suggested that there are substantial postprandial differences in rumen fermentation variables and microbiota between samples collected using a stomach tube versus those obtained via rumen cannulation techniques (
      • de Assis Lage C.F.
      • Raisanen S.E.
      • Melgar A.
      • Nedelkov K.
      • Chen X.J.
      • Oh J.
      • Fetter M.E.
      • Indugu N.
      • Bender J.S.
      • Vecchiarelli B.
      • Hennessy M.L.
      • Pitta D.
      • Hristov A.N.
      Comparison of two sampling techniques for evaluating ruminal fermentation and microbiota in the planktonic phase of rumen digesta in dairy cows.
      ;
      • Hagey J.V.
      • Laabs M.
      • Maga E.A.
      • DePeters E.J.
      Rumen sampling methods bias bacterial communities observed.
      ). However, rumen liquid collected via a stomach tube is a routinely used method for rumen sample collection because of its simplicity (
      • Lodge-Ivey S.L.
      • Browne-Silva J.
      • Horvath M.B.
      Bacterial diversity and fermentation end products in rumen fluid samples collected via oral lavage or rumen cannula.
      ;
      • Henderson G.
      • Cox F.
      • Kittelmann S.
      • Miri V.H.
      • Zethof M.
      • Noel S.J.
      • Waghorn G.C.
      • Janssen P.H.
      Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
      ;
      • Ramos-Morales E.
      • Arco-Pérez A.
      • Martín-García A.I.
      • Yánez-Ruiz D.R.
      • Frutos P.
      • Hervás G.
      Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats.
      ). In terms of rumen microbiome and fermentation parameters, rumen fluid extracted via the fistula has been shown to be comparable to fluid extracted via an oral stomach tube (
      • Paz H.A.
      • Anderson C.L.
      • Muller M.J.
      • Kononoff P.J.
      • Fernando S.C.
      Rumen bacterial community composition in Holstein and Jersey cows is different under same dietary condition and is not affected by sampling method.
      ;
      • Song J.
      • Choi H.
      • Jeong J.Y.
      • Lee S.
      • Lee H.J.
      • Baek Y.
      • Ji S.Y.
      • Kim M.
      Effects of sampling techniques and sites on rumen microbiome and fermentation parameters in Hanwoo steers.
      ). Rumen bacteria diversity was mainly affected by diet and cow individuals, and using different fractions of rumen content to assess bacterial diversity will generate similar results (
      • Ji S.K.
      • Zhang H.T.
      • Yan H.
      • Azarfar A.
      • Shi H.T.
      • Alugongo G.
      • Li S.L.
      • Cao Z.J.
      • Wang Y.J.
      Comparison of rumen bacteria distribution in original rumen digesta, rumen liquid and solid fractions in lactating Holstein cows.
      ).
      Moreover, discarding the first 200 mL of rumen fluid may help minimize saliva contamination, potentially altering fermentation parameters. However, an effect of saliva on rumen pH may still exist (
      • de Assis Lage C.F.
      • Raisanen S.E.
      • Melgar A.
      • Nedelkov K.
      • Chen X.J.
      • Oh J.
      • Fetter M.E.
      • Indugu N.
      • Bender J.S.
      • Vecchiarelli B.
      • Hennessy M.L.
      • Pitta D.
      • Hristov A.N.
      Comparison of two sampling techniques for evaluating ruminal fermentation and microbiota in the planktonic phase of rumen digesta in dairy cows.
      ), so we did not present rumen pH results in this study, even though noninvasive stomach tubing is a feasible alternative to surgical rumen cannulation in dairy cows to examine ruminal fermentation and microbiome. Nonetheless, caution should be used when using this technique to assess the structure and composition of the rumen microbial community in future studies. In this study, based on the data presented following collection via a stomach tube, the replacement of soybean meal by HOPSC did not have a significant effect on rumen fermentation parameters. Therefore, HOPSC and DDGS can partially or completely replace soybean meal in diets of high-yielding dairy cows without affecting performance under the conditions of this experiment.

      Rumen Bacterial Composition

      Rumen microorganisms are complex and functionally diverse, directly affecting dairy cows' production performance; therefore, the effect of new feed ingredients on rumen microorganisms is one of the landmark indicators for evaluating their feed value (
      • Matthews C.
      • Crispie F.
      • Lewis E.
      • Reid M.
      • O’Toole P.W.
      • Cotter P.D.
      The rumen microbiome: A crucial consideration when optimising milk and meat production and nitrogen utilisation efficiency.
      ). In this study, although the fat content in the diet increased with HOPSC, the dietary fat content did not exceed the safe range (5%), even with complete replacement of soybean meal, and theoretically, it would not pose any health risk to dairy cows (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ). In this study, we found that rumen microorganisms still changed slightly. The feeding value of HOPSC was further assessed by analyzing α-diversity indexes of ruminal bacteria (Table 4). The ACE, Chao 1, Shannon, and Simpson indexes did not differ when HOPSC replaced soybean meal, indicating that short-term replacement of soybean meal with HOPSC did not influence the richness or diversity of rumen bacteria in dairy cows.
      Considering the rumen bacteria profile at the phylum and genus levels, an increase in dietary fat content resulted in a balanced and steady state, as a whole, and only part of the rumen bacterial abundance changed. Previous studies have shown that Firmicutes and Bacteroidetes change independent of caloric intake (
      • Turnbaugh P.J.
      • Ley R.E.
      • Mahowald M.A.
      • Magrini V.
      • Mardis E.R.
      • Gordon J.I.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      ), so the changes in this study may not be related to increased dietary energy. Rather, it could be that UFA had a coating effect on the feed particles in the rumen. As part of the feed, feed particles could be covered by UFA, which might impede fermentation and result in rumen microbial changes (
      • Jenkins T.C.
      Lipid metabolism in the rumen.
      ). Second, fatty acids can destroy bacterial cell membranes and be toxic to rumen microorganisms, leading to changes in rumen microorganisms (
      • Jenkins T.C.
      Lipid metabolism in the rumen.
      ). Another reason could be related to the addition of DDGS to the diet. Studies have shown that replacing soybean meal with DDGS can cause changes in rumen microorganisms, with an increase in the abundance of Firmicutes and Tenericutes and a decrease in the population of Bacteroidetes (
      • Castillo-Lopez E.
      • Jenkins C.J.R.
      • Aluthge N.D.
      • Tom W.
      • Kononoff P.J.
      • Fernando S.C.
      The effect of regular or reduced-fat distillers grains with solubles on rumen methanogenesis and the rumen bacterial community.
      ), which is consistent with the results of this study. The trend of Tenericutes is also consistent with that of DDGS addition, but the effect of the increase in UFA cannot be ruled out, and other factors may need to be further studied. The abundance of Prevotella also decreased, consistent with the change in Bacteroidetes, and rumen Ruminococcus showed an opposite trend to Firmicutes. However, the study showed that the replacement of soybean meal with DDGS did not cause a decrease in the abundance of Ruminococcus, so the changes in the abundance of Ruminococcus in this study were not related to DDGS (
      • Castillo-Lopez E.
      • Jenkins C.J.R.
      • Aluthge N.D.
      • Tom W.
      • Kononoff P.J.
      • Fernando S.C.
      The effect of regular or reduced-fat distillers grains with solubles on rumen methanogenesis and the rumen bacterial community.
      ). Ruminococcus is a fibrinolytic bacteria, which can be inhibited under a certain concentration of UFA and has high sensitivity to UFA (
      • Maia M.R.G.
      • Chaudhary L.C.
      • Figueres L.
      • Wallace R.J.
      Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen.
      ). The decrease in abundance of Ruminococcus may be caused by dietary intake of UFA. Therefore, it is necessary to pay attention to the number and activity of fibrolytic bacteria to avoid a negative effect on fiber degradation in the rumen when regulating the concentration of UFA in the diet. In this experiment, the α-diversity results indicated that overall species diversity was always in a stable state. However, in terms of β-diversity, PCA and PLS-DA highlighted the changes in rumen microorganisms after HOPSC replaced soybean meal. The PCA can project OTU data of the sample flora into the spatial coordinate system through linear transformation to reveal the natural distribution of the sample. Compared with PCA, PLS-DA is more suitable for small intergroup differences and can better emphasize intergroup differences (
      • Lasalvia M.
      • Capozzi V.
      • Perna G.
      A comparison of PCA-LDA and PLS-DA techniques for classification of vibrational spectra.
      ). Both analytical methods led to the same conclusion; the rumen microflora of cows in the 100HOPSC group differed from that of the control group. Such changes cannot be more directly reflected through short-term experiments, and whether the direction of change is beneficial or harmful is unknown and requires a long-term study to verify.

      Milk Fatty Acid Composition

      Some studies have found that the fatty acid composition in milk can be regulated by changing the dietary composition of dairy cows or supplementing additional vegetable oil (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ;
      • Welter K.C.
      • Martins C.M.M.R.
      • de Palma A.S.V.
      • Martins M.M.
      • Dos Reis B.R.
      • Schmidt B.L.U.
      • Saran Netto A.
      Canola oil in lactating dairy cow diets reduces milk saturated fatty acids and improves its omega-3 and oleic fatty acid content.
      ). However, our results showed that the replacement of soybean meal with different proportions of HOPSC did not cause changes in milk fatty acids, which may be caused by the hydrogenation of microorganisms (
      • Maia M.R.G.
      • Chaudhary L.C.
      • Bestwick C.S.
      • Richardson A.J.
      • McKain N.
      • Larson T.R.
      • Graham I.A.
      • Wallace R.J.
      Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens..
      ). It is believed that UFA are harmful to rumen microorganisms, which will eventually convert most of the UFA into SFA through hydrogenation, resulting in poor fluidity of UFA. Still, only a small proportion of PUFA finds its way into meat and milk, and HOPSC did not successfully regulate the fatty acids in milk (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ). Regulating fatty acids in cow milk by changing the diet or adding fat supplements has always been challenging.
      Factors such as ruminant species, lactation period, and external environment affect the fatty acid composition in milk and the complex fatty acid metabolism pathway in dairy cows. Most of the oleic acid and linoleic acid in the HOPSC is hydrogenated by microorganisms after entering the rumen, which will produce C18 and various isomers of MUFA and PUFA; the products of different diets are different. These products are absorbed by the intestinal tract and used for various purposes; some are directly passed into the milk and some are converted by the mammary gland tissue, making the results of regulation difficult to judge (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ).
      • Klir Z.
      • Castro-Montoya J.M.
      • Novoselec J.
      • Molkentin J.
      • Domacinovic M.
      • Mioc B.
      • Dickhoefer U.
      • Antunovic Z.
      Influence of pumpkin seed cake and extruded linseed on milk production and milk fatty acid profile in Alpine goats.
      used pumpkin seed cake to completely replace soybean meal in dairy goat feed and found that it had little effect on the fatty acid content of dairy goat milk, similar to the results of this experiment.
      • Šalavardić Ž.K.
      • Novoselec J.
      • Castro-Montoya J.M.
      • Šperanda M.
      • Đidara M.
      • Molkentin J.
      • Mioč B.
      • Dickhoefer U.
      • Antunović Z.
      The effect of dietary pumpkin seed cake and extruded linseed on blood haemato-chemicals and milk quality in Alpine goats during early lactation.
      also used pumpkin seed cake to completely replace soybean meal to feed Alpine goats during early lactation and found that it had little effect on the fatty acid content in milk, proving the complexity of regulating fatty acids. In addition, muscle fatty acids are difficult to regulate due to the complex metabolic pathways of fatty acids.
      • Antunović Z.
      • Klir Ž.
      • Šperanda M.
      • Sičaja V.
      • Čolović D.
      • Mioč B.
      • Novoselec J.
      Partial replacement of soybean meal with pumpkin seed cake in lamb diets: Effects on carcass traits, haemato-chemical parameters and fatty acids in meat.
      fed lambs with pumpkin seed cake to replace 10% and 15% soybean meal and found little difference in muscle fatty acid concentrations in semimembranosus muscle. Therefore, more detailed research needs to be done using different breeds, lactation periods, and diets to regulate fatty acids in dairy cows.

      CONCLUSIONS

      This work serves as a preliminary study on the replacement of soybean meal with HOPSC. Our findings on lactation performance, rumen fermentation parameters, and rumen bacteria show that a high proportion of HOPSC can replace soybean meal without affecting the production performance of dairy cows. Also, due to the increasing content of UFA in the rumen, the hydrogenation process of rumen microorganisms was increased. Whether long-term feeding could affect changes in rumen microbial abundance, fatty acid partitioning between adipose tissue and mammary glands requires further validation. Still, it is clear that short-term feeding of HOPSC as an alternative to soybean meal is not detrimental. The production performance, rumen environment, and bacterial community remain stable, effectively reducing the feeding cost and serving as a potential solution to the shortage of high-quality protein feed.

      ACKNOWLEDGMENTS

      This study was financially supported by the earmarked fund for CARS36 (Beijing, China). The authors have not stated any conflicts of interest.

      REFERENCES

        • Antunović Z.
        • Klir Ž.
        • Šperanda M.
        • Sičaja V.
        • Čolović D.
        • Mioč B.
        • Novoselec J.
        Partial replacement of soybean meal with pumpkin seed cake in lamb diets: Effects on carcass traits, haemato-chemical parameters and fatty acids in meat.
        S. Afr. J. Anim. Sci. 2018; 48: 695-704
        • AOAC International
        Official Methods of Analysis.
        16th ed. AOAC International, 2005
        • Bardaa S.
        • Ben Halima N.
        • Aloui F.
        • Ben Mansour R.
        • Jabeur H.
        • Bouaziz M.
        • Sahnoun Z.
        Oil from pumpkin (Cucurbita pepo L.) seeds: Evaluation of its functional properties on wound healing in rats.
        Lipids Health Dis. 2016; 15 (27068642): 73
        • Bayat A.R.
        • Vilkki J.
        • Razzaghi A.
        • Leskinen H.
        • Kettunen H.
        • Khurana R.
        • Brand T.
        • Ahvenjärvi S.
        Evaluating the effects of high-oil rapeseed cake or natural additives on methane emissions and performance of dairy cows.
        J. Dairy Sci. 2022; 105 (34799103): 1211-1224
        • Bionaz M.
        • Vargas-Bello-Pérez E.
        • Busato S.
        Advances in fatty acids nutrition in dairy cows: From gut to cells and effects on performance.
        J. Anim. Sci. Biotechnol. 2020; 11 (33292523): 110
        • Broderick G.A.
        • Kang J.H.
        Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media.
        J. Dairy Sci. 1980; 63 (7372898): 64-75
        • Cammack K.M.
        • Austin K.J.
        • Lamberson W.R.
        • Conant G.C.
        • Cunningham H.C.
        Ruminant Nutrition Symposium: Tiny but mighty: The role of the rumen microbes in livestock production.
        J. Anim. Sci. 2018; 96 (30272229): 752-770
        • Caporaso J.G.
        • Kuczynski J.
        • Stombaugh J.
        • Bittinger K.
        • Bushman F.D.
        • Costello E.K.
        • Fierer N.
        • Peña A.
        • Goodrich J.K.
        • Gordon J.I.
        • Huttley G.A.
        • Kelley S.T.
        • Knights D.
        • Koenig J.E.
        • Ley R.E.
        • Lozupone C.A.
        • McDonald D.
        • Muegge B.D.
        • Pirrung M.
        • Reeder J.
        • Sevinsky J.R.
        • Turnbaugh P.J.
        • Walters W.A.
        • Widmann J.
        • Yatsunenko T.
        • Zaneveld J.
        • Knight R.
        QIIME allows analysis of high-throughput community sequencing data.
        Nat. Methods. 2010; 7 (20383131): 335-336
        • Castillo-Lopez E.
        • Jenkins C.J.R.
        • Aluthge N.D.
        • Tom W.
        • Kononoff P.J.
        • Fernando S.C.
        The effect of regular or reduced-fat distillers grains with solubles on rumen methanogenesis and the rumen bacterial community.
        J. Appl. Microbiol. 2017; 123 (28891118): 1381-1395
        • Chilliard Y.
        • Glasser F.
        • Ferlay A.
        • Bernard L.
        • Rouel J.
        • Doreau M.
        Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
        Eur. J. Lipid Sci. Technol. 2007; 109: 828-855
        • de Assis Lage C.F.
        • Raisanen S.E.
        • Melgar A.
        • Nedelkov K.
        • Chen X.J.
        • Oh J.
        • Fetter M.E.
        • Indugu N.
        • Bender J.S.
        • Vecchiarelli B.
        • Hennessy M.L.
        • Pitta D.
        • Hristov A.N.
        Comparison of two sampling techniques for evaluating ruminal fermentation and microbiota in the planktonic phase of rumen digesta in dairy cows.
        Front. Microbiol. 2020; 11 (33424820)618032
        • Enjalbert F.
        • Combes S.
        • Zened A.
        • Meynadier A.
        Rumen microbiota and dietary fat: A mutual shaping.
        J. Appl. Microbiol. 2017; 123 (28557277): 782-797
        • Gidlund H.
        • Hetta M.
        • Krizsan S.J.
        • Lemosquet S.
        • Huhtanen P.
        Effects of soybean meal or canola meal on milk production and methane emissions in lactating dairy cows fed grass silage-based diets.
        J. Dairy Sci. 2015; 98 (26364100): 8093-8106
        • Hagey J.V.
        • Laabs M.
        • Maga E.A.
        • DePeters E.J.
        Rumen sampling methods bias bacterial communities observed.
        PLoS One. 2022; 17 (35511785)e0258176
        • Hao X.Y.
        • Yu S.C.
        • Mu C.T.
        • Wu X.D.
        • Zhang C.X.
        • Zhao J.X.
        • Zhang J.X.
        Replacing soybean meal with flax seed meal: Effects on nutrient digestibility, rumen microbial protein synthesis and growth performance in sheep.
        Animal. 2020; 14 (32172723): 1841-1848
        • Henderson G.
        • Cox F.
        • Kittelmann S.
        • Miri V.H.
        • Zethof M.
        • Noel S.J.
        • Waghorn G.C.
        • Janssen P.H.
        Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
        PLoS One. 2013; 8 (24040342)e74787
        • Jenkins T.C.
        Lipid metabolism in the rumen.
        J. Dairy Sci. 1993; 76 (8132891): 3851-3863
        • Ji S.K.
        • Zhang H.T.
        • Yan H.
        • Azarfar A.
        • Shi H.T.
        • Alugongo G.
        • Li S.L.
        • Cao Z.J.
        • Wang Y.J.
        Comparison of rumen bacteria distribution in original rumen digesta, rumen liquid and solid fractions in lactating Holstein cows.
        J. Anim. Sci. Biotechnol. 2017; 8 (28168037): 16
        • Jiang X.
        • Xu H.J.
        • Ma G.M.
        • Sun Y.K.
        • Li Y.
        • Zhang Y.G.
        Digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows fed a fermented corn gluten-wheat bran mixture as a substitute for soybean meal.
        J. Dairy Sci. 2021; 104 (33455755): 2866-2880
        • Klir Z.
        • Castro-Montoya J.M.
        • Novoselec J.
        • Molkentin J.
        • Domacinovic M.
        • Mioc B.
        • Dickhoefer U.
        • Antunovic Z.
        Influence of pumpkin seed cake and extruded linseed on milk production and milk fatty acid profile in Alpine goats.
        Animal. 2017; 11 (28367773): 1772-1778
        • Lasalvia M.
        • Capozzi V.
        • Perna G.
        A comparison of PCA-LDA and PLS-DA techniques for classification of vibrational spectra.
        Appl. Sci. (Basel). 2022; 125345
        • Li Y.
        • Wu Q.H.
        • Lv J.Y.
        • Jia X.M.
        • Gao J.X.
        • Zhang Y.G.
        • Wang L.
        Associations of protein molecular structures with their nutrient supply and biodegradation characteristics in different byproducts of seed-used pumpkin.
        Animals (Basel). 2022; 12 (35454203): 956
        • Li Y.
        • Zhang G.N.
        • Fang X.P.
        • Zhao C.
        • Wu H.Y.
        • Lan Y.X.
        • Che L.
        • Sun Y.K.
        • Lv J.Y.
        • Zhang Y.G.
        • Pan C.F.
        Effects of replacing soybean meal with pumpkin seed cake and dried distillers grains with solubles on milk performance and antioxidant functions in dairy cows.
        Animal. 2021; 15 (33526406)100004
        • Lodge-Ivey S.L.
        • Browne-Silva J.
        • Horvath M.B.
        Bacterial diversity and fermentation end products in rumen fluid samples collected via oral lavage or rumen cannula.
        J. Anim. Sci. 2009; 87 (19329475): 2333-2337
        • Loor J.J.
        • Elolimy A.A.
        • Mccann J.C.
        Dietary impacts on rumen microbiota in beef and dairy production.
        Anim. Front. 2016; 6: 22-29
        • Lopes J.C.
        • Harper M.T.
        • Giallongo F.
        • Oh J.
        • Smith L.
        • Ortega-Perez A.M.
        • Harper S.A.
        • Melgar A.
        • Kniffen D.M.
        • Fabin R.A.
        • Hristov A.N.
        Effect of high-oleic-acid soybeans on production performance, milk fatty acid composition, and enteric methane emission in dairy cows.
        J. Dairy Sci. 2017; 100 (27988126): 1122-1135
        • Maia M.R.G.
        • Chaudhary L.C.
        • Bestwick C.S.
        • Richardson A.J.
        • McKain N.
        • Larson T.R.
        • Graham I.A.
        • Wallace R.J.
        Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens..
        BMC Microbiol. 2010; 10 (20167098): 52
        • Maia M.R.G.
        • Chaudhary L.C.
        • Figueres L.
        • Wallace R.J.
        Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen.
        Antonie van Leeuwenhoek. 2007; 91 (17072533): 303-314
        • Mao S.Y.
        • Zhang M.L.
        • Liu J.H.
        • Zhu W.Y.
        Characterising the bacterial microbiota across the gastrointestinal tracts of dairy cattle: Membership and potential function.
        Sci. Rep. 2015; 5 (26527325)16116
        • Matthews C.
        • Crispie F.
        • Lewis E.
        • Reid M.
        • O’Toole P.W.
        • Cotter P.D.
        The rumen microbiome: A crucial consideration when optimising milk and meat production and nitrogen utilisation efficiency.
        Gut Microbes. 2019; 10 (30207838): 115-132
        • Mele M.
        • Buccioni A.
        • Serra A.
        • Antongiovanni M.
        • Secchiari P.
        Lipids of goat’s milk: Origin, composition and main sources of variation.
        in: Cannas A. Pulina G. Dairy Goats Feeding and Nutrition. CAB International, 2008: 47-70
        • Mozaffarian D.
        • Katan M.B.
        • Ascherio A.
        • Stampfer M.J.
        • Willett W.C.
        Trans fatty acids and cardiovascular disease.
        N. Engl. J. Med. 2006; 354 (16611951): 1601-1613
        • NRC
        Nutrient Requirements of Dairy Cattle.
        7th rev. ed. National Academies Press, 2001
        • Paula E.M.
        • Broderick G.A.
        • Danes M.A.C.
        • Lobos N.E.
        • Zanton G.I.
        • Faciola A.P.
        Effects of replacing soybean meal with canola meal or treated canola meal on ruminal digestion, omasal nutrient flow, and performance in lactating dairy cows.
        J. Dairy Sci. 2018; 101 (29129322): 328-339
        • Paz H.A.
        • Anderson C.L.
        • Muller M.J.
        • Kononoff P.J.
        • Fernando S.C.
        Rumen bacterial community composition in Holstein and Jersey cows is different under same dietary condition and is not affected by sampling method.
        Front. Microbiol. 2016; 7 (27536291)1206
        • Ramos-Morales E.
        • Arco-Pérez A.
        • Martín-García A.I.
        • Yánez-Ruiz D.R.
        • Frutos P.
        • Hervás G.
        Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats.
        Anim. Feed Sci. Technol. 2014; 198: 57-66
        • Šalavardić Ž.K.
        • Novoselec J.
        • Castro-Montoya J.M.
        • Šperanda M.
        • Đidara M.
        • Molkentin J.
        • Mioč B.
        • Dickhoefer U.
        • Antunović Z.
        The effect of dietary pumpkin seed cake and extruded linseed on blood haemato-chemicals and milk quality in Alpine goats during early lactation.
        Mljekarstvo. 2021; 71: 13-24
        • Sezgin A.
        • Aydın B.
        Effect of replacing dietary soybean meal with pumpkin (Cucurbita pepo) seed cake on growth, feed utilization, haematological parameters and fatty acid composition of mirror carp (Cyprinus carpio).
        Aquacult. Res. 2021; 52: 5870-5881
        • Shingfield K.J.
        • Ahvenjärvi S.
        • Toivonen V.
        • Ärölä A.
        • Nurmela K.V.V.
        • Huhtanen P.
        • Griinari J.M.
        Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows.
        Anim. Sci. 2003; 77: 165-179
        • Siri-Tarino P.W.
        • Sun Q.
        • Hu F.B.
        • Krauss R.M.
        Saturated fat, carbohydrate, and cardiovascular disease.
        Am. J. Clin. Nutr. 2010; 91 (20089734): 502-509
        • Song J.
        • Choi H.
        • Jeong J.Y.
        • Lee S.
        • Lee H.J.
        • Baek Y.
        • Ji S.Y.
        • Kim M.
        Effects of sampling techniques and sites on rumen microbiome and fermentation parameters in Hanwoo steers.
        J. Microbiol. Biotechnol. 2018; 28 (29996593): 1700-1705
        • Stewart C.S.
        • Duncan S.H.
        The effect of avoparcin on cellulolytic bacteria of the ovine rumen.
        Microbiology (Reading). 1985; 131: 427-435
        • Tedeschi L.O.
        • Chalupa W.
        • Janczewski E.
        • Fox D.G.
        • Sniffen C.J.
        • Munson R.
        • Kononoff P.J.
        • Boston R.C.
        Evaluation and application of the CPM Dairy nutrition model.
        J. Agric. Sci. 2008; 146: 171-182
        • Turnbaugh P.J.
        • Ley R.E.
        • Mahowald M.A.
        • Magrini V.
        • Mardis E.R.
        • Gordon J.I.
        An obesity-associated gut microbiome with increased capacity for energy harvest.
        Nature. 2006; 444 (17183312): 1027-1031
        • Van Soest P.J.
        • Robertson J.B.
        • Lewis B.A.
        Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
        J. Dairy Sci. 1991; 74 (1660498): 3583-3597
        • Vargas-Bello-Pérez E.
        • Cancino-Padilla N.
        • Romero J.
        • Garnsworthy P.C.
        Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils.
        Animal. 2016; 10 (27146195): 1821-1828
        • Wei Z.
        • Zhang B.
        • Liu J.
        Effects of the dietary nonfiber carbohydrate content on lactation performance, rumen fermentation, and nitrogen utilization in mid-lactation dairy cows receiving corn stover.
        J. Anim. Sci. Biotechnol. 2018; 9: 1-7
        • Welter K.C.
        • Martins C.M.M.R.
        • de Palma A.S.V.
        • Martins M.M.
        • Dos Reis B.R.
        • Schmidt B.L.U.
        • Saran Netto A.
        Canola oil in lactating dairy cow diets reduces milk saturated fatty acids and improves its omega-3 and oleic fatty acid content.
        PLoS One. 2016; 11 (27015405)e0151876
        • Wolff S.M.
        • Ellison M.J.
        • Hao Y.
        • Cockrum R.R.
        • Austin K.J.
        • Baraboo M.
        • Burch K.
        • Lee H.J.
        • Maurer T.
        • Patil R.D.
        • Ravelo A.
        • Taxis T.M.
        • Truong H.
        • Lamberson W.R.
        • Cammack K.M.
        • Conant G.C.
        Diet shifts provoke complex and variable changes in the metabolic networks of the ruminal microbiome.
        Microbiome. 2017; 5 (28595639): 60
        • Zdunczyk Z.
        • Minakowski D.
        • Frejnagel S.
        • Flis M.
        Comparative study of the chemical composition and nutritional value of pumpkin seed cake, soybean meal and casein.
        Nahrung. 1999; 43 (10633538): 392-395