Reprod Dom Anim 50, 735–739 (2015); doi: 10.1111/rda.12566 ISSN 0936–6768
Reproductive and Metabolic Responses of Early-lactating Dairy Cows Fed Different Dietary Protein Sources V Tufarelli, GM Lacalandra and V Laudadio Section of Veterinary Science and Animal Production, Department of Emergency and Organ Transplantation (DETO), University of Bari ‘Aldo Moro’, Valenzano, Bari, Italy
Contents Optimal reproduction is very closely tied with optimal nutrition, and early-lactation diets in cows are critical to successful reproduction and monitoring is important. To evaluate the effects of different dietary protein sources on metabolic parameters and reproductive activity, a total of 36 Italian Friesian early-lactating dairy cows were assigned for 16 weeks to three dietary treatments as follow: the control diet contained soya bean meal (SBM) as the main protein source, whereas the experimental diets contained faba bean (FB) or pea seeds (PS) as alternative protein sources. Diets were formulated to be isocaloric and isonitrogenous. Cow blood samples were collected, and plasma were analysed for metabolites, biological enzymes, b-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA). Feeding alternative protein sources had no effects on most metabolic blood profile, except for blood cholesterol, triglycerides and urea. Results from reproductive parameters indicated that cows fed FB diet had a lower insemination index, but a shorter calving to conception period and an improved conception rate and artificial insemination outcome, when compared to cows fed SBM or PS diets. It can be concluded that replacing conventional dietary SBM with alternative protein sources, especially FB, resulted in improved reproductive performances and metabolic parameters in early-lactating dairy cows.
Introduction Nutrition has an important impact on the reproductive performance of dairy and beef cattle. The impact of nutrition on fertility of dairy herds may be caused by direct and indirect effects of nutrients on reproductive traits. Productive performances per dairy cow have increased consistently over the past decades with a concomitant decrease in conception rate and increase in health problems. Milk production has been reported to be greater for cows that ovulate sooner post-partum (Staples et al. 1990) and have fewer days open (Emanuelson and Oltenacu 1998). Farms with greater milk production have been associated with higher incidence of diseases such as clinical mastitis, cystic ovaries and silent oestrus but the increased reporting of treating of these diseases may imply better management on farms. As nutritionists, an increasing challenge will be to formulate diets based on the latest information that promote reproductive health, thus dietary proteins are nutrients selected to discuss in terms of their potential impact on fertility (Santos 2001).
© 2015 Blackwell Verlag GmbH
Among dietary protein sources, research has clearly shown that legumes can increase growth rates in ruminants due to higher nitrogen utilisation efficiency and dry matter intake (Speijers et al. 2004; Marley et al. 2007; Chand et al. 2014). Grain legumes are feedstuffs of high protein and metabolizable energy content (McCarthy et al. 2015). They are readily accepted by cattle, and animals require little adaptation to avoid rumen acidosis and reduced intakes. Legumes are likely to be particularly useful as supplements for low quality feeds in feeding systems where low levels of management input make it difficult to ensure satisfactory adaptation to cereal-based supplements (Tufarelli et al. 2012). However, one constraint to the use of legume-based cow systems is the presence of phytooestrogen compounds present in the forage reduces cow fertility. Phyto-oestrogens are a group of naturally occurring plant-derived non-steroidal compounds, which have the ability to cause estrogenic and/or anti-estrogenic effects in livestock species (Benassayag et al. 2002). On the other hand, the different plant species contain different types and also different concentrations of these oestrogen-mimicking compounds, which may have led to contrary anecdotal evidence as to the effects of legumes per se on ruminant fertility. So, there is a need to develop clear messages on the effects of these forage legumes on ruminant fertility for the farming industry, messages which consider the availability of new varieties, current understanding and future research requirements to overcome the practical constraints for their use in dairy farms. Following a review of the available literature, no evidence of research into the effect of legume grains on fertility in cows was evidenced. Previous research by Marley et al. (2007) on lupin grain investigated the effects of feeding concentrates containing either yellow lupins or soya bean to mature dairy cows, and it was found indicated that lupins could play a role in replacing imported soya bean in the diet of dairy cows in the many countries, but that further research is necessary to determine the effects of other legume grains on cows fertility. Therefore, this study was planned to provide alternative feeding strategies for dairy farming comparing the diets based on different protein sources (soya bean, pea and faba bean) to assess their effect on and metabolic and reproductive responses of early-lactating dairy cows.
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Materials and Methods Animals and diets The present trial was conducted in a dairy farm, located in Bari Province of Apulia region in Southern Italy, for 18 weeks and involved 36 multiparous Holstein dairy cows in early stage of lactation. The research included a 2-week adaptation period to the diet followed by 16 weeks of feeding the three experimental diets. The cows [(625 21 kg body weight (BW); 2.6 0.3 in body condition score (BCS); 21 5 days in milk (DIM) at the beginning of the trial)] were cared for according to the animal welfare requirements. Cows were individually housed in pens during the adaptation period and in tie-stalls during the experimental period. Each stall had a separate manger for hay feeding and was equipped with a watering outlet. Wood shavings were used as bedding materials. Cows had free access to fresh water during the whole trial. Cow health status was checked throughout the study, and no cases of clinical mastitis were recorded. Cows (n = 12 per treatment) were randomly allocated to three dietary treatments according to calving date, parity and their milk yield and composition. The dietary treatments were as follows: (i) a control concentrate, which contained soya bean meal (SBM; 150 g/kg) as the main proteinous feed; (ii) an experimental concentrate containing faba bean (FB; 345 g/ kg); and (iii) an experimental concentrate containing pea seeds (PS; 375 g/kg); as the main proteinous feed in substitution of SBM (Table 1). The three concentrates contained 170 g/kg crude protein (CP) as reported in Table 2. The FB used in the trial were locally and organically grown (Vicia faba L. var minor, cv. Prothabat 69), contained 261 g/kg CP (% DM basis) and estimated 8.55 MJ net energy for lactation (NEL)/kg DM; whereas dietary PS utilized were also organically and locally grown (Pisun sativum L. cv. Spirale) containing 251 g/kg CP and an estimated 7.57 MJ/kg of NEL. The protein feeds were processed and coarsely ground at a commercial feedstuff factory. The diet offered to the all groups consisted of ad libitum oat hay supplemented with SBM, PS and FB concentrates. Hay and concentrates were daily given in three meals (06.00, 12.00 and 18.00 h). The concentrate levels were adjusted on a weekly basis on cows’ milk yield. The quantity of concentrate was always totally eaten by each cow, and no concentrate refusals were recorded during the whole experimental period. Individual hay intake was measured by decreasing the daily dose offered to each cow by the residue recovered in the manger. The diets fed in this trial were formulated according to NRC (2001) to provide similar amounts of NEL, CP and met but did not exceed the nutrient requirements of a Holstein cow yielding 35 kg milk/day with 3.5% milk fat and 3.0% milk protein. The energy values for lactation (UFL) of the hay and concentrates were estimated (Jarrige 1989). The reproductive activity of
V Tufarelli, GM Lacalandra and V Laudadio Table 1. Ingredient composition of experimental concentrates fed to dairy cows Dietary treatment Item
SBM
FB
PS
Ingredient, % Ground corn Durum wheat bran Soya bean meal Faba bean Pea seeds Sunflower meal Dehydrated beet pulp Corn gluten feed Calcium carbonate Dicalcium phosphate Sodium chloride Sodium bicarbonate Vitamin-mineral premixa Magnesium oxide Yeast
345.0 249.0 150.0 – – 75.0 75.0 50.0 24.0 10.0 5.5 5.5 5.0 3.5 2.5
195.0 224.0 – 345.0 – 75.0 75.0 55.0 50.0 24.0 10.0 5.5 5.5 5.0 3.5
125.0 244.0 – – 375.0 75.0 75.0 50.0 24.0 10.0 5.5 5.5 5.0 3.5 2.5
SBM, soya bean meal; FB, faba bean; PS, pea seeds. a Supplied per kg of diet: vitamin A 40,000 IU; vitamin D3 4,000 IU; vitamin E 60 mg; vitamin B1 10 mg; vitamin B3 500 mg; choline chloride 250 mg; vitamin B12 0.03 mg; Co 1.25 mg; Fe 100 mg; I 5 mg; Mn 100 mg; Cu 20 mg; Se 0.25 mg; Zn 215 mg.
Table 2. Chemical composition (% on dry matter basis) of diets fed to dairy cows Dietary treatment Item
SBM
FB
PS
Oat hay
Chemical composition, % Dry matter Crude protein Ether extract Crude fibre Ash NDF ADF Lignin Starch NFC Acid insoluble ash Ca P Na NEL, Mcal/kg DM Milk FU, n/kg DM
88.65 16.97 2.67 7.46 8.15 21.13 9.22 2.37 29.88 51.08 0.89 1.38 0.70 0.43 1.39 0.89
88.11 16.91 2.49 7.71 8.21 22.54 9.87 2.58 28.21 49.85 0.95 1.39 0.71 0.42 1.38 0.89
89.02 16.32 2.52 7.79 8.07 21.87 9.69 2.67 29.58 48.78 1.01 1.38 0.71 0.42 1.38 0.88
89.87 6.35 2.04 29.77 9.29 53.44 31.85 3.32 28.05 1.09 0.38 0.15 0.02 0.53 0.51
SBM, soya bean meal; FB, faba bean; PS, pea seeds; NDF, neutral detergent fibre; ADF, acid detergent fibre; NFC, non-fibre carbohydrates. NFC = 100 – (% NDF + % crude protein + % ether extract + % ash). NEL, Net Energy for Lactation (NRC, 2001). Milk FU, Feed Unit for Lactation (INRA, 1989).
each cow was recorded, with particular care to clinical and reproductive parameters as follows: the presence of abnormal cycle, the calving-conceiving period, the insemination index, intercalving period and the ovarian steroids levels. Artificial insemination was made after a voluntary waiting period of 60 days. © 2015 Blackwell Verlag GmbH
Dietary Protein Source and Reproduction in Cow
Feeds sampling and analysis Samples of hay, concentrates and refusals were daily collected and concentrate feeds were weekly sampled. Samples were ground in an hammer mill provided with a 1-mm pore size screen and analysed in triplicate for their content in DM (forced-air oven at 65°C and dried to a constant weight), ash, CP (N 9 6.25), crude fibre (CF), ether extract (EE), Ca, P and Na according to the procedures outlined by the AOAC (2000). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were analysed according to Van Soest et al. (1991) and corrected for residual acid insoluble ash (AIA). Lignin was determined by the method of Van Soest and Robertson (1985). Starch was measured after acid hydrolysis and polarimetric detection according to Holm et al. (1986). Non-fibre carbohydrates (NFC) percentages were calculated as follows: 100 (NDF + CP + EE + ash). Blood sampling, analysis and hormonal assay Cow’s blood samples were collected every 2 weeks, prior to morning feeding via coccygeal venipuncture and immediately chilled. Plasma samples, obtained after centrifugation at 2000 g 9 20 min, were stored at 20°C until the analysis. Stored samples were determined using commercial kits (Sentinel Chemical, Milan, Italy) and an automatic spectrophotometer (Cobas FARA 2; Roche Diagnostic, Basel, Switzerland) for total protein, albumin, glucose, total cholesterol, blood urea (BU), triglycerides, non-esterified fatty acids (NEFA), calcium, inorganic phosphorus and b-hydroxybutyrate (BHBA). Globulin content was calculated as the difference between total protein and albumin. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed by commercial kits produced by Bio-Merieux (Marcy-l’Etoile, France) and Boehringer Mannheim (Maylan, France), following the methodology suggested by the producers. Plasma P4 and E2 concentrations were measured (at 0, 30, 60 and 90 days) using the Enzyme-Linked Fluorescent Assay (ELFA) kits (VIDASÒ Progesterone and VIDASÒ Estradiol II, SYSMEX bioMerieux, Tokyo, Japan, respectively), which were validated for bovine plasma (Anckaert et al. 2002; Hossam et al. 2013). The current assays showed high correlation to the reference method (radioimmunoassay; RIA). Moreover, the curve resulting from serial dilution of bovine plasma was parallel to the standard curve. The intra- and interassay coefficients of variation for the P4 and E2 assays agreed the validation of the kit (Anckaert et al. 2002). Statistical analysis The experimental design was a randomized block design, with 12 replicates per treatment. Data were analysed as repeated measures with diet as a non© 2015 Blackwell Verlag GmbH
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repeated factor using analysis of variance using the GLM procedure of SAS (2004). Sources of variation included cows and dietary treatment. Cows within treatment were fitted as term of error. When significant effect was found, means were compared using Student’s t-test. Significant differences were declared at p < 0.05 and a trend at p < 0.10. All results were expressed as the mean SEM, unless otherwise stated.
Results and Discussion The three concentrates fed to early-lactating cows had similar levels of crude protein, crude fibre, NEL as well as total fibre and its fractions; however, both pea and FB diets had slightly lower NFC with similar starch content, which reflects the difference in starch content between corn grain and legumes (Table 2). The comparison between our results and previous trials must be carried out with caution because of earlier studies conducted on cows were performed using a combination of other leguminous grains such as FB, pea, lupin and clover (Froidmon and Bartiaux-Thill 2004; Vander Pol et al. 2008, Cozzi et al. 2010; Tufarelli et al. 2012; Volpelli et al. 2009). To the best our knowledge, the present trial is one of the few studies in the literature describing the effect of the total replacement of SBM with peas and FB on metabolic and reproductive responses in dairy cows. Consequently, data on the influence of peas and FB on the reproduction activities of cows in the early phase of lactation are quite limited. No significant difference occurred in most of evaluated blood metabolites, electrolytes or enzymes between control-soya bean and experimental pea and FB diets (Table 3). In particular, it was observed a significant change in the content of total cholesterol and triglyceride in blood, with the higher (p = 0.021) cholesterol values of 237.7 mg/dl in the blood samples of cows fed soya bean, against 216.4 and 210.3 mg/dl in FB and pea, respectively. In the same way, the blood levels of triglycerides were higher (p = 0.039; 6.3 mg/dl) in cows fed with soya bean when compared to cows fed diets containing FB and pea (6.1 and 5.8 mg/dl, respectively). Moreover, in our study, the BU concentration recorded in cows fed experimental diets were reduced significantly, especially in pea diet, and it was almost certainly due to a decreased degradability of pea protein leading to a decreased ammonia level in the rumen and urea level in blood, as also reported in a recent study of Volpelli et al. (2009). The effect of dietary protein sources on reproductive parameters of early-lactating dairy cows is reported in Table 4. Regarding to cows reproductive aspects, in all dietary groups we found a good calving-first heat period, calving-conceiving period and intercalving period. In particular, the cows fed diet containing FB showed a low index of insemination (p = 0.041), but a high positive outcome of artificial insemination and a conception rate equal to 100% when compared to cows fed diets containing pea or soya bean, which showed
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V Tufarelli, GM Lacalandra and V Laudadio 8.0
Table 3. Effect of dietary protein sources on metabolic biochemical blood profile of dairy cows
Item
SBM
FB
PS
SEM
p-Value
Total protein, g/dl Albumin, g/dl Globulin, g/dl Calcium, mg/dl Phosphorus, mg/dl Total cholesterol, mg/dl Triglycerides, mg/dl Blood urea, mg/dl Glucose, mg/dl AST, IU/l ALT, IU/l NEFA, mmol/l BHBA, lmol/l
7.6 3.4 4.5 8.5 5.6 237.7 6.3 36.2 48.7 86.2 42.4 0.3 548.8
7.6 3.5 4.1 8.9 6.1 216.4 6.1 33.1 50.3 85.1 37.7 0.3 554.4
7.5 3.5 4.0 8.5 5.8 210.3 5.8 32.2 46.4 87.7 38.5 0.3 575.1
0.05 0.02 0.07 0.09 0.13 4.01 0.15 0.42 0.25 0.33 0.69 0.02 6.78
0.451 0.362 0.470 0.111 0.513 0.021 0.039 0.042 0.198 0.087 0.070 0.366 0.075
P4 (ng/ml)
Dietary treatment
Item
SBM
FB
PS
SEM
39.0
23.7
24.1
4.36
133.5
99.2
115.3
1.5 2 (16%)
1.3 12 (100%)
1.7 10 (83%)
p-Value
PS
5.0 4.0 3.0
1.0 0.0
0
30
60
90
Days
Fig. 1. Plasma progesterone (P4) concentrations (ng/ml) of dairy cows (n = 12 per treatment) fed the experimental diets. SBM, soya bean meal; FB, faba bean; PS, pea seeds
E2 (pg/ml)
Dietary treatment
FB
6.0
2.0
SBM, soya bean meal; FB, faba bean; PS, pea seeds. AST, aspartate aminotransferase; ALT, alanine aminotransferase; NEFA, nonesterified fatty acids; BHBA; b-hydroxybutyrate.
Table 4. Effect of dietary protein sources on reproductive parameters of dairy cows (n = 12 per treatment)
SBM
7.0
16.0
SBM
15.0
FB
14.0
PS
13.0 12.0 11.0 10.0
Calving to 1st heat, days Calving to conception, days Insemination index Artificial insemination outcome, n Conception rate, %
70
100
58
17.7
0.023 0.032
0.05 –
0.041 0.015
–
0.012
9.0 8.0
0
30
60
90
Days
Fig. 2. Plasma estradiol (E2, 17-b-estradiol) concentrations (pg/ml) of dairy cows (n = 12 per treatment) the experimental diets. SBM, soya bean meal; FB, faba bean; PS, pea seeds
SBM, soya bean meal; FB, faba bean; PS, pea seeds.
conception rates of 58 and 70%, respectively (p = 0.012). Furthermore, our results show that cows fed diets containing FB reported an artificial insemination outcome of 100%, compared to 16 and 83% in animals fed soya bean and pea diets, respectively (p = 0.015). Feeding field bean diet has also significantly (p = 0.032) reduced the cows calving to conception period being equal to 108.3 days compared to 133.5 and 115.3 days in soya bean and pea dietary groups, respectively. As reported in Fig. 1, in early-lactating cows fed with peas and FBs the progesteronemia increased gradually until it reaches levels consistent with the state of pregnancy clinically diagnosed in cows, resulting therefore in levels of 17-b-estradiol undetectable or very low (Fig. 2). On the other hand, dairy cows fed with diets based on soya bean showed the lowest values of progesteronemia associated with higher plasma estradiol oestrogen levels. Thus, based on these findings, it is possible to state that the luteal and ovarian activity in early-lactating cows resulted improved in those fed the alternative dietary protein sources as peas and FB.
Errors in diet formulation (excesses and deficiencies) are often the most important cause of bovine hypofertility (Walsh et al. 2011). Soya bean is a dietary protein source characterized by high biological value, particularly for its content of lysine and tryptophan as well as methionine and cysteine; therefore, soya bean represents a good feed especially for livestock species (Laudadio and Tufarelli 2011; Tufarelli et al. 2012; Khan et al. 2014). However, soya bean is one of the most genetically modified plants containing a considerable amount of phytoestrogens, structurally and functionally similar to 17-b-estradiol, represented by isoflavones including genistein and daidzein (Burton and Wells 2002; Woclawek-Potocka et al. 2005; Tufarelli et al. 2015). Soya bean replacement with FB or PS may be thus effective, removing also the risk of transgenic components in diet. A lower content of phytoestrogens and the related modifications of the endocrine balance confirmed a significant improvement of cow’s reproductive performances. The incidence of related pathologies is reduced, extending the productive and reproductive cow’s career and letting down the recovery rate, follicular cysts and the repeat breeder syndrome (Bertoni et al. 2009; Sartori © 2015 Blackwell Verlag GmbH
Dietary Protein Source and Reproduction in Cow
et al. 2009; Yusuf et al. 2010). The positive cow’s metabolic variations, particularly in the hepatic values, observed in the present trial, allow us to consider positively the soya bean replacement with the alternative dietary protein sources. In conclusion, this study demonstrated that replacing conventional dietary SBM with alternative protein sources, especially FB, resulted in improved reproductive performances and metabolic parameters in earlylactating dairy cows.
References Anckaert E, Mees M, Schiettecatte J, Smitz J, 2002: Clinical validation of a fully automated 17b-estradiol and progesterone assay (VIDASÒ) for use in monitoring assisted reproduction treatment. Clin Chem Lab Med 40, 824–831. AOAC, 2000: Official Methods of Analysis, 17th edn. Association of Official Analytical Chemists, Gaithersburg, MD. Benassayag C, Perrot-Applanat M, Ferre F, 2002: Phytoestrogens as modulators of steroid action in target cells. J Chromatogr B Analyt Technol Biomed Life Sci 777, 233–248. Bertoni G, Trevisi E, Lombardelli R, 2009: Some new aspects of nutrition, health conditions and fertility of intensively reared dairy cows. Ital J Anim Sci 8, 491–518. Burton JL, Wells M, 2002: The effect of phytoestrogens on the female genital tract. J Clin Pathol 55, 401–407. Chand N, Ihsanuddin, Khan RU, 2014: Replacement of soybean meal with yeast single cell protein in broiler ration: the effect on performance traits. Pak J Zool 46, 1753–1758. Cozzi G, Boukh A, Contiero B, Gottardo F, 2010: Extruded pea (Pisum sativum) as alternative to soybean protein for dairy cows feeding in organic Alpine farms. Ital J Anim Sci 9, 194–199. Emanuelson U, Oltenacu PA, 1998: Incidences and effects of diseases on the performance of Swedish dairy herds stratified by production. J Dairy Sci 81, 2376– 2382. Froidmon E, Bartiaux-Thill N, 2004: Suitability of lupin and pea seeds as a substitute for soybean meal in high-producing dairy cow feed. Anim Res 53, 475– 487. Holm J, Bj€ orck I, Drews A, Asp NG, 1986: A rapid method for the analysis of starch. Starch/St€ arke 38, 224–226. Hossam EL, Kitahara G, Tamura Y, Kobayashi I, Hemmi K, Torisu S, Sameshima H, Horii Y, Zaabel S, Kamimura S, 2013: Presence of a temperature gradient among genital tract portions and the thermal changes within these portions over the estrous cycle in beef cows. J Reprod Dev 59, 59–65.
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Conflict of interest The authors declare that there is no any conflict of interest.
Author contributions All authors conceived and coordinated the study, conducted statistical analysis, interpreted the results and drafted the manuscript. All authors read and approved the manuscript.
INRA, 1989: Ruminant Nutrition. Recommended Allowances and Feed Tables. INRA, Paris, France. Jarrige R, 1989: Ruminant Nutrition: Ruminant Nutrition Recommended Allowances and Feed Tables. INRA, Versailles. Khan RU, Zia-ur-Rahman, Javed I, Muhammad F, 2014: Serum antioxidants and trace minerals as influenced by vitamins, probiotics and proteins in broiler breeders. J Appl Anim Res 42, 249–255. Laudadio V, Tufarelli V, 2011: Dehulledmicronised lupin (Lupinus albus L. cv. Multitalia) as the main protein source for broilers: influence on growth performance, carcass traits and meat fatty acid composition. J Sci Food Agric 91, 2081– 2087. Marley CL, Fychan R, Fraser MD, Sanderson R, Jones R, 2007: Effects of feeding different ensiled forages on the productivity and nutrient-use efficiency of finishing lambs. Grass Forage Sci 62, 1–12. McCarthy MM, Yasui T, Ryan CM, Mechor GD, Overton TR, 2015: Performance of early-lactation dairy cows as affected by dietary starch and monensin supplementation. J Dairy Sci 98, 3335– 3350. NRC, 2001: Nutrient Requirements of Dairy Cattle, 7th revised edn. National Academy Press, Washington, DC. Santos JE, 2001: Dietary ingredients and nutritional management impact fertility in dairy cattle. Proc. of the 36th Annual Pacific Northwest Animal Nutrition Conference, pp. 189–220. Sartori R, Bastos MR, Wiltbank MC, 2009: Factors affecting fertilisation and early embryo quality in single-and superovulated dairy cattle. Reprod Fertil Develop 22, 151–158. SAS, Statistical Analysis System, 2004: SAS/ STAT. User’s Guide. SAS Inst, Cary, NC. Speijers MHM, Fraser MD, Theobald VJ, Haresign W, 2004: The effects of grazing forage legumes on the performance of finishing lambs. J Agric Sci 142, 483–493. Staples CR, Thatcher WW, Clark JH, 1990: Relationship between ovarian activity and energy status during the early postpartum period of high producing dairy cows. J Dairy Sci 73, 938–947. Tufarelli V, Khan RU, Laudadio V, 2012: Evaluating the suitability of field beans as a substitute for soybean meal in early-
lactating dairy cow: Production and metabolic responses. Anim Sci J 83, 136–140. Tufarelli V, Selvaggi M, Dario C, Laudadio V, 2015: Genetically modified feeds in poultry diet: safety, performance, and product quality. Crit Rev Food Sci Nutr 55, 562–569. Van Soest PJ, Robertson JB, 1985: Analysis of Forages and Fibrous Feeds. Laboratory Manual for Animal Science 613. Cornell University, Ithaca, NY. Van Soest PJ, Robertson JB, Lewis BA, 1991: Methods for dietary fiber, neutral detergent fiber, and non-starch olysaccharides in relation to animal nutrition. J Dairy Sci 4, 3583–3597. Vander Pol M, Hristov AN, Zaman S, Delano N, 2008: Peas can replace soybean meal and corn grain in dairy cow diets. J Dairy Sci 91, 698–703. Volpelli A, Comellini M, Masoero F, Moschini M, Lo Fiego DP, Scipioni R, 2009: Pea (Pisum sativum) in dairy cow diet: effect on milk production and quality. Ital J Anim Sci 8, 245–257. Walsh SW, Williams EJ, Evans ACO, 2011: A review of the causes of poor fertility in high milk producing dairy cows. Anim Reprod Sci 123, 127–138. Woclawek-Potocka I, Acosta T, Korzekwa A, Bah MM, Shibaya M, Okuda K, Skarzynski DJ, 2005: Phytoestrogens modulate prostaglandin production in bovine endometrium specificity and intracellular mechanisms. Exp Biol Med (Maywood) 230, 326–333. Yusuf M, Nakao T, Ranasinghe RBK, Gautam G, Long ST, Yoshida C, Koike K, Hayashi A, 2010: Reproductive performance of repeat breeders in dairy herds. Theriogenology 73, 1220–1229.
Submitted: 22 May 2015; Accepted: 6 Jun 2015 Author’s address (for correspondence): V Tufarelli, Section of Veterinary Science and Animal Production, Department of Emergency and Organ Transplantation (DETO), University of Bari ‘Aldo Moro’, Strada Prov. le per Casamassima km 3, 70010 Valenzano (BA), Italy. E-mail: vincenzo. [email protected]
Reproductive and Metabolic Responses of Early-lactating Dairy Cows Fed Different Dietary Protein Sources V Tufarelli, GM Lacalandra and V Laudadio Section of Veterinary Science and Animal Production, Department of Emergency and Organ Transplantation (DETO), University of Bari ‘Aldo Moro’, Valenzano, Bari, Italy
Contents Optimal reproduction is very closely tied with optimal nutrition, and early-lactation diets in cows are critical to successful reproduction and monitoring is important. To evaluate the effects of different dietary protein sources on metabolic parameters and reproductive activity, a total of 36 Italian Friesian early-lactating dairy cows were assigned for 16 weeks to three dietary treatments as follow: the control diet contained soya bean meal (SBM) as the main protein source, whereas the experimental diets contained faba bean (FB) or pea seeds (PS) as alternative protein sources. Diets were formulated to be isocaloric and isonitrogenous. Cow blood samples were collected, and plasma were analysed for metabolites, biological enzymes, b-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA). Feeding alternative protein sources had no effects on most metabolic blood profile, except for blood cholesterol, triglycerides and urea. Results from reproductive parameters indicated that cows fed FB diet had a lower insemination index, but a shorter calving to conception period and an improved conception rate and artificial insemination outcome, when compared to cows fed SBM or PS diets. It can be concluded that replacing conventional dietary SBM with alternative protein sources, especially FB, resulted in improved reproductive performances and metabolic parameters in early-lactating dairy cows.
Introduction Nutrition has an important impact on the reproductive performance of dairy and beef cattle. The impact of nutrition on fertility of dairy herds may be caused by direct and indirect effects of nutrients on reproductive traits. Productive performances per dairy cow have increased consistently over the past decades with a concomitant decrease in conception rate and increase in health problems. Milk production has been reported to be greater for cows that ovulate sooner post-partum (Staples et al. 1990) and have fewer days open (Emanuelson and Oltenacu 1998). Farms with greater milk production have been associated with higher incidence of diseases such as clinical mastitis, cystic ovaries and silent oestrus but the increased reporting of treating of these diseases may imply better management on farms. As nutritionists, an increasing challenge will be to formulate diets based on the latest information that promote reproductive health, thus dietary proteins are nutrients selected to discuss in terms of their potential impact on fertility (Santos 2001).
© 2015 Blackwell Verlag GmbH
Among dietary protein sources, research has clearly shown that legumes can increase growth rates in ruminants due to higher nitrogen utilisation efficiency and dry matter intake (Speijers et al. 2004; Marley et al. 2007; Chand et al. 2014). Grain legumes are feedstuffs of high protein and metabolizable energy content (McCarthy et al. 2015). They are readily accepted by cattle, and animals require little adaptation to avoid rumen acidosis and reduced intakes. Legumes are likely to be particularly useful as supplements for low quality feeds in feeding systems where low levels of management input make it difficult to ensure satisfactory adaptation to cereal-based supplements (Tufarelli et al. 2012). However, one constraint to the use of legume-based cow systems is the presence of phytooestrogen compounds present in the forage reduces cow fertility. Phyto-oestrogens are a group of naturally occurring plant-derived non-steroidal compounds, which have the ability to cause estrogenic and/or anti-estrogenic effects in livestock species (Benassayag et al. 2002). On the other hand, the different plant species contain different types and also different concentrations of these oestrogen-mimicking compounds, which may have led to contrary anecdotal evidence as to the effects of legumes per se on ruminant fertility. So, there is a need to develop clear messages on the effects of these forage legumes on ruminant fertility for the farming industry, messages which consider the availability of new varieties, current understanding and future research requirements to overcome the practical constraints for their use in dairy farms. Following a review of the available literature, no evidence of research into the effect of legume grains on fertility in cows was evidenced. Previous research by Marley et al. (2007) on lupin grain investigated the effects of feeding concentrates containing either yellow lupins or soya bean to mature dairy cows, and it was found indicated that lupins could play a role in replacing imported soya bean in the diet of dairy cows in the many countries, but that further research is necessary to determine the effects of other legume grains on cows fertility. Therefore, this study was planned to provide alternative feeding strategies for dairy farming comparing the diets based on different protein sources (soya bean, pea and faba bean) to assess their effect on and metabolic and reproductive responses of early-lactating dairy cows.
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Materials and Methods Animals and diets The present trial was conducted in a dairy farm, located in Bari Province of Apulia region in Southern Italy, for 18 weeks and involved 36 multiparous Holstein dairy cows in early stage of lactation. The research included a 2-week adaptation period to the diet followed by 16 weeks of feeding the three experimental diets. The cows [(625 21 kg body weight (BW); 2.6 0.3 in body condition score (BCS); 21 5 days in milk (DIM) at the beginning of the trial)] were cared for according to the animal welfare requirements. Cows were individually housed in pens during the adaptation period and in tie-stalls during the experimental period. Each stall had a separate manger for hay feeding and was equipped with a watering outlet. Wood shavings were used as bedding materials. Cows had free access to fresh water during the whole trial. Cow health status was checked throughout the study, and no cases of clinical mastitis were recorded. Cows (n = 12 per treatment) were randomly allocated to three dietary treatments according to calving date, parity and their milk yield and composition. The dietary treatments were as follows: (i) a control concentrate, which contained soya bean meal (SBM; 150 g/kg) as the main proteinous feed; (ii) an experimental concentrate containing faba bean (FB; 345 g/ kg); and (iii) an experimental concentrate containing pea seeds (PS; 375 g/kg); as the main proteinous feed in substitution of SBM (Table 1). The three concentrates contained 170 g/kg crude protein (CP) as reported in Table 2. The FB used in the trial were locally and organically grown (Vicia faba L. var minor, cv. Prothabat 69), contained 261 g/kg CP (% DM basis) and estimated 8.55 MJ net energy for lactation (NEL)/kg DM; whereas dietary PS utilized were also organically and locally grown (Pisun sativum L. cv. Spirale) containing 251 g/kg CP and an estimated 7.57 MJ/kg of NEL. The protein feeds were processed and coarsely ground at a commercial feedstuff factory. The diet offered to the all groups consisted of ad libitum oat hay supplemented with SBM, PS and FB concentrates. Hay and concentrates were daily given in three meals (06.00, 12.00 and 18.00 h). The concentrate levels were adjusted on a weekly basis on cows’ milk yield. The quantity of concentrate was always totally eaten by each cow, and no concentrate refusals were recorded during the whole experimental period. Individual hay intake was measured by decreasing the daily dose offered to each cow by the residue recovered in the manger. The diets fed in this trial were formulated according to NRC (2001) to provide similar amounts of NEL, CP and met but did not exceed the nutrient requirements of a Holstein cow yielding 35 kg milk/day with 3.5% milk fat and 3.0% milk protein. The energy values for lactation (UFL) of the hay and concentrates were estimated (Jarrige 1989). The reproductive activity of
V Tufarelli, GM Lacalandra and V Laudadio Table 1. Ingredient composition of experimental concentrates fed to dairy cows Dietary treatment Item
SBM
FB
PS
Ingredient, % Ground corn Durum wheat bran Soya bean meal Faba bean Pea seeds Sunflower meal Dehydrated beet pulp Corn gluten feed Calcium carbonate Dicalcium phosphate Sodium chloride Sodium bicarbonate Vitamin-mineral premixa Magnesium oxide Yeast
345.0 249.0 150.0 – – 75.0 75.0 50.0 24.0 10.0 5.5 5.5 5.0 3.5 2.5
195.0 224.0 – 345.0 – 75.0 75.0 55.0 50.0 24.0 10.0 5.5 5.5 5.0 3.5
125.0 244.0 – – 375.0 75.0 75.0 50.0 24.0 10.0 5.5 5.5 5.0 3.5 2.5
SBM, soya bean meal; FB, faba bean; PS, pea seeds. a Supplied per kg of diet: vitamin A 40,000 IU; vitamin D3 4,000 IU; vitamin E 60 mg; vitamin B1 10 mg; vitamin B3 500 mg; choline chloride 250 mg; vitamin B12 0.03 mg; Co 1.25 mg; Fe 100 mg; I 5 mg; Mn 100 mg; Cu 20 mg; Se 0.25 mg; Zn 215 mg.
Table 2. Chemical composition (% on dry matter basis) of diets fed to dairy cows Dietary treatment Item
SBM
FB
PS
Oat hay
Chemical composition, % Dry matter Crude protein Ether extract Crude fibre Ash NDF ADF Lignin Starch NFC Acid insoluble ash Ca P Na NEL, Mcal/kg DM Milk FU, n/kg DM
88.65 16.97 2.67 7.46 8.15 21.13 9.22 2.37 29.88 51.08 0.89 1.38 0.70 0.43 1.39 0.89
88.11 16.91 2.49 7.71 8.21 22.54 9.87 2.58 28.21 49.85 0.95 1.39 0.71 0.42 1.38 0.89
89.02 16.32 2.52 7.79 8.07 21.87 9.69 2.67 29.58 48.78 1.01 1.38 0.71 0.42 1.38 0.88
89.87 6.35 2.04 29.77 9.29 53.44 31.85 3.32 28.05 1.09 0.38 0.15 0.02 0.53 0.51
SBM, soya bean meal; FB, faba bean; PS, pea seeds; NDF, neutral detergent fibre; ADF, acid detergent fibre; NFC, non-fibre carbohydrates. NFC = 100 – (% NDF + % crude protein + % ether extract + % ash). NEL, Net Energy for Lactation (NRC, 2001). Milk FU, Feed Unit for Lactation (INRA, 1989).
each cow was recorded, with particular care to clinical and reproductive parameters as follows: the presence of abnormal cycle, the calving-conceiving period, the insemination index, intercalving period and the ovarian steroids levels. Artificial insemination was made after a voluntary waiting period of 60 days. © 2015 Blackwell Verlag GmbH
Dietary Protein Source and Reproduction in Cow
Feeds sampling and analysis Samples of hay, concentrates and refusals were daily collected and concentrate feeds were weekly sampled. Samples were ground in an hammer mill provided with a 1-mm pore size screen and analysed in triplicate for their content in DM (forced-air oven at 65°C and dried to a constant weight), ash, CP (N 9 6.25), crude fibre (CF), ether extract (EE), Ca, P and Na according to the procedures outlined by the AOAC (2000). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were analysed according to Van Soest et al. (1991) and corrected for residual acid insoluble ash (AIA). Lignin was determined by the method of Van Soest and Robertson (1985). Starch was measured after acid hydrolysis and polarimetric detection according to Holm et al. (1986). Non-fibre carbohydrates (NFC) percentages were calculated as follows: 100 (NDF + CP + EE + ash). Blood sampling, analysis and hormonal assay Cow’s blood samples were collected every 2 weeks, prior to morning feeding via coccygeal venipuncture and immediately chilled. Plasma samples, obtained after centrifugation at 2000 g 9 20 min, were stored at 20°C until the analysis. Stored samples were determined using commercial kits (Sentinel Chemical, Milan, Italy) and an automatic spectrophotometer (Cobas FARA 2; Roche Diagnostic, Basel, Switzerland) for total protein, albumin, glucose, total cholesterol, blood urea (BU), triglycerides, non-esterified fatty acids (NEFA), calcium, inorganic phosphorus and b-hydroxybutyrate (BHBA). Globulin content was calculated as the difference between total protein and albumin. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed by commercial kits produced by Bio-Merieux (Marcy-l’Etoile, France) and Boehringer Mannheim (Maylan, France), following the methodology suggested by the producers. Plasma P4 and E2 concentrations were measured (at 0, 30, 60 and 90 days) using the Enzyme-Linked Fluorescent Assay (ELFA) kits (VIDASÒ Progesterone and VIDASÒ Estradiol II, SYSMEX bioMerieux, Tokyo, Japan, respectively), which were validated for bovine plasma (Anckaert et al. 2002; Hossam et al. 2013). The current assays showed high correlation to the reference method (radioimmunoassay; RIA). Moreover, the curve resulting from serial dilution of bovine plasma was parallel to the standard curve. The intra- and interassay coefficients of variation for the P4 and E2 assays agreed the validation of the kit (Anckaert et al. 2002). Statistical analysis The experimental design was a randomized block design, with 12 replicates per treatment. Data were analysed as repeated measures with diet as a non© 2015 Blackwell Verlag GmbH
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repeated factor using analysis of variance using the GLM procedure of SAS (2004). Sources of variation included cows and dietary treatment. Cows within treatment were fitted as term of error. When significant effect was found, means were compared using Student’s t-test. Significant differences were declared at p < 0.05 and a trend at p < 0.10. All results were expressed as the mean SEM, unless otherwise stated.
Results and Discussion The three concentrates fed to early-lactating cows had similar levels of crude protein, crude fibre, NEL as well as total fibre and its fractions; however, both pea and FB diets had slightly lower NFC with similar starch content, which reflects the difference in starch content between corn grain and legumes (Table 2). The comparison between our results and previous trials must be carried out with caution because of earlier studies conducted on cows were performed using a combination of other leguminous grains such as FB, pea, lupin and clover (Froidmon and Bartiaux-Thill 2004; Vander Pol et al. 2008, Cozzi et al. 2010; Tufarelli et al. 2012; Volpelli et al. 2009). To the best our knowledge, the present trial is one of the few studies in the literature describing the effect of the total replacement of SBM with peas and FB on metabolic and reproductive responses in dairy cows. Consequently, data on the influence of peas and FB on the reproduction activities of cows in the early phase of lactation are quite limited. No significant difference occurred in most of evaluated blood metabolites, electrolytes or enzymes between control-soya bean and experimental pea and FB diets (Table 3). In particular, it was observed a significant change in the content of total cholesterol and triglyceride in blood, with the higher (p = 0.021) cholesterol values of 237.7 mg/dl in the blood samples of cows fed soya bean, against 216.4 and 210.3 mg/dl in FB and pea, respectively. In the same way, the blood levels of triglycerides were higher (p = 0.039; 6.3 mg/dl) in cows fed with soya bean when compared to cows fed diets containing FB and pea (6.1 and 5.8 mg/dl, respectively). Moreover, in our study, the BU concentration recorded in cows fed experimental diets were reduced significantly, especially in pea diet, and it was almost certainly due to a decreased degradability of pea protein leading to a decreased ammonia level in the rumen and urea level in blood, as also reported in a recent study of Volpelli et al. (2009). The effect of dietary protein sources on reproductive parameters of early-lactating dairy cows is reported in Table 4. Regarding to cows reproductive aspects, in all dietary groups we found a good calving-first heat period, calving-conceiving period and intercalving period. In particular, the cows fed diet containing FB showed a low index of insemination (p = 0.041), but a high positive outcome of artificial insemination and a conception rate equal to 100% when compared to cows fed diets containing pea or soya bean, which showed
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V Tufarelli, GM Lacalandra and V Laudadio 8.0
Table 3. Effect of dietary protein sources on metabolic biochemical blood profile of dairy cows
Item
SBM
FB
PS
SEM
p-Value
Total protein, g/dl Albumin, g/dl Globulin, g/dl Calcium, mg/dl Phosphorus, mg/dl Total cholesterol, mg/dl Triglycerides, mg/dl Blood urea, mg/dl Glucose, mg/dl AST, IU/l ALT, IU/l NEFA, mmol/l BHBA, lmol/l
7.6 3.4 4.5 8.5 5.6 237.7 6.3 36.2 48.7 86.2 42.4 0.3 548.8
7.6 3.5 4.1 8.9 6.1 216.4 6.1 33.1 50.3 85.1 37.7 0.3 554.4
7.5 3.5 4.0 8.5 5.8 210.3 5.8 32.2 46.4 87.7 38.5 0.3 575.1
0.05 0.02 0.07 0.09 0.13 4.01 0.15 0.42 0.25 0.33 0.69 0.02 6.78
0.451 0.362 0.470 0.111 0.513 0.021 0.039 0.042 0.198 0.087 0.070 0.366 0.075
P4 (ng/ml)
Dietary treatment
Item
SBM
FB
PS
SEM
39.0
23.7
24.1
4.36
133.5
99.2
115.3
1.5 2 (16%)
1.3 12 (100%)
1.7 10 (83%)
p-Value
PS
5.0 4.0 3.0
1.0 0.0
0
30
60
90
Days
Fig. 1. Plasma progesterone (P4) concentrations (ng/ml) of dairy cows (n = 12 per treatment) fed the experimental diets. SBM, soya bean meal; FB, faba bean; PS, pea seeds
E2 (pg/ml)
Dietary treatment
FB
6.0
2.0
SBM, soya bean meal; FB, faba bean; PS, pea seeds. AST, aspartate aminotransferase; ALT, alanine aminotransferase; NEFA, nonesterified fatty acids; BHBA; b-hydroxybutyrate.
Table 4. Effect of dietary protein sources on reproductive parameters of dairy cows (n = 12 per treatment)
SBM
7.0
16.0
SBM
15.0
FB
14.0
PS
13.0 12.0 11.0 10.0
Calving to 1st heat, days Calving to conception, days Insemination index Artificial insemination outcome, n Conception rate, %
70
100
58
17.7
0.023 0.032
0.05 –
0.041 0.015
–
0.012
9.0 8.0
0
30
60
90
Days
Fig. 2. Plasma estradiol (E2, 17-b-estradiol) concentrations (pg/ml) of dairy cows (n = 12 per treatment) the experimental diets. SBM, soya bean meal; FB, faba bean; PS, pea seeds
SBM, soya bean meal; FB, faba bean; PS, pea seeds.
conception rates of 58 and 70%, respectively (p = 0.012). Furthermore, our results show that cows fed diets containing FB reported an artificial insemination outcome of 100%, compared to 16 and 83% in animals fed soya bean and pea diets, respectively (p = 0.015). Feeding field bean diet has also significantly (p = 0.032) reduced the cows calving to conception period being equal to 108.3 days compared to 133.5 and 115.3 days in soya bean and pea dietary groups, respectively. As reported in Fig. 1, in early-lactating cows fed with peas and FBs the progesteronemia increased gradually until it reaches levels consistent with the state of pregnancy clinically diagnosed in cows, resulting therefore in levels of 17-b-estradiol undetectable or very low (Fig. 2). On the other hand, dairy cows fed with diets based on soya bean showed the lowest values of progesteronemia associated with higher plasma estradiol oestrogen levels. Thus, based on these findings, it is possible to state that the luteal and ovarian activity in early-lactating cows resulted improved in those fed the alternative dietary protein sources as peas and FB.
Errors in diet formulation (excesses and deficiencies) are often the most important cause of bovine hypofertility (Walsh et al. 2011). Soya bean is a dietary protein source characterized by high biological value, particularly for its content of lysine and tryptophan as well as methionine and cysteine; therefore, soya bean represents a good feed especially for livestock species (Laudadio and Tufarelli 2011; Tufarelli et al. 2012; Khan et al. 2014). However, soya bean is one of the most genetically modified plants containing a considerable amount of phytoestrogens, structurally and functionally similar to 17-b-estradiol, represented by isoflavones including genistein and daidzein (Burton and Wells 2002; Woclawek-Potocka et al. 2005; Tufarelli et al. 2015). Soya bean replacement with FB or PS may be thus effective, removing also the risk of transgenic components in diet. A lower content of phytoestrogens and the related modifications of the endocrine balance confirmed a significant improvement of cow’s reproductive performances. The incidence of related pathologies is reduced, extending the productive and reproductive cow’s career and letting down the recovery rate, follicular cysts and the repeat breeder syndrome (Bertoni et al. 2009; Sartori © 2015 Blackwell Verlag GmbH
Dietary Protein Source and Reproduction in Cow
et al. 2009; Yusuf et al. 2010). The positive cow’s metabolic variations, particularly in the hepatic values, observed in the present trial, allow us to consider positively the soya bean replacement with the alternative dietary protein sources. In conclusion, this study demonstrated that replacing conventional dietary SBM with alternative protein sources, especially FB, resulted in improved reproductive performances and metabolic parameters in earlylactating dairy cows.
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Conflict of interest The authors declare that there is no any conflict of interest.
Author contributions All authors conceived and coordinated the study, conducted statistical analysis, interpreted the results and drafted the manuscript. All authors read and approved the manuscript.
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Submitted: 22 May 2015; Accepted: 6 Jun 2015 Author’s address (for correspondence): V Tufarelli, Section of Veterinary Science and Animal Production, Department of Emergency and Organ Transplantation (DETO), University of Bari ‘Aldo Moro’, Strada Prov. le per Casamassima km 3, 70010 Valenzano (BA), Italy. E-mail: vincenzo. [email protected]
