Effect of low protein diets supplemented with rumen protected Methionine, Lysine and Choline on Holstein dairy cows productive and reproductive performance

Abstract
Introduction: Genetic selection for the higher milk yield during the last decades, resulted in lower fertility in dairy cattle herds (Dobson et al. 2007; Walsh et al. 2011). There are handful reports that the efficient fertility and higher production in dairy cattle requires greater concentration of nutrients such as protein, energy and micronutrients in the diet (Gao et al. 2009; Roche et al. 2011; Bisinotto et al. 2018; Ayyat et al. 2021). The dietary crude protein (CP) has vital role both in milk yield and fertility rate, however it has not received enough attention (Ayyat et al. 2021; Cardoso et al. 2021). Dietary crude protein (CP), can be degraded in the rumen (RDP) to prepare nitrogen (N) for ruminal microorganisms to produce microbial protein at the presence of carbohydrates or not to be degraded in the rumen (RUP), but digested by intestinal enzymes to prepare amino acids for host animal (Kamalak et al. 2005; Taghizadeh et al. 2005). Increasing CP level to provide enough quantities of RDP and RUP may lead to an excessive N in the blood (BUN) and milk (MUN), as the monitors of the dietary protein both in quality and quantity (Hammon et al. 2005). Surplus level of RDP will result in higher concentration of ruminal ammonia (De Boever et al. 2005) and subsequently urea, a product of detoxification of ammonia by liver (Tamminga 2006). These metabolic residues have adverse effects on reproduction and fertility, where ammonia interferes prior to the ovulation, whereas urea has detrimental effects after fertilization (Sinclair et al. 2000; Jorritsma et al. 2003). Hence, limiting CP level may be a reasonable strategy to control consequences, but on the other hand, it has been reported that reducing CP level may decrease metabolizable protein (MP; RUP+ by pass microbial protein) which will result in the reduction of limiting amino acids (NRC 2001). Methionine (Met) and lysine (Lys) are first limiting amino acids in the diets based on corn (NRC 2001). Supplementing diets with rumen protected (RP) Met can enhance dairy cattle performance and dry matter intake (DMI), also it can boost liver function by reducing accumulation of the lipid (Ordway et al. 2009; Osorio et al. 2013). Methionine was proposed as limiting amino acid in the reproduction trait of dairy cattle (Métayer et al. 2008), where a correlation of Met concentration and embryonic development was reported by Acosta et al. (2016) beside its concentration in embryonic and uterine fluids (Groebner et al. 2011). Moreover, Met and Lys are participated in taurine and glutathione synthesis, where glutathione is involved in Glutathione transferase, Glutathione peroxidase and Glutathione reductase as cellular detoxifiers (Mavrommatis et al. 2021). Methionine is reported to be methyl donor in the synthesis of carnitine and choline (Chandler and White 2017). Choline (Chol) may be a key component in the synthesis of phosphatidylcholine, acetylcholine and as a methyl donor like Met. It has been shown that Chol has an important role in lipid metabolism consequently the energy balance (Esposito et al. 2014).
Therefore, the objective of this study was to decrease CP level in order to reduce unsuitable effects of suppressing N on dairy cattle reproduction trait and to compensate for amino acid loss, rumen protected Met, Lys and Chol were added as top dress to the diets.

Material method: Thirty multiparous and thirty primiparous (n=60) Holstein dairy cows with the BCS (body condition score) of 3 were individually housed in free-stall barns. Cows were used in two periods of 28 days as experimental animals. All animals of each period (30 cows; 15 multiparous and 15 primiparous) were treated according to Iranian Council of Animal Care guide lines (1995). Cows were randomly assigned to experimental diets based on their parity, days in milk (DIM) and milk yield. Designed five experimental diets were (1) Control diet (CP=16.2; providing +149 g/d of MP requirements according to NRC (2001); (2) Low protein diet (LPD) +methionine (LPDM; CP=14.2; −232 g/d of MP requirements + 30 gr/cow per day of RPMet [Mepron, Evonik Nutrition & Care GmbH, Hanau, Germany); (3) LPD +lysine (LPDL; CP=14.2; −232 g/d of MP requirements,+ 100 gr/cow per day RPLys of [RELys, VETAGRO S.p.A.; Reggio Emilia, Italy]); (4) LPD +methionine + lysine (LPDML; CP=14.2); (5) LPD +methionine + lysine + choline (LPDMLC; CP=14.2; + 60 gr/cow per day of RPChol, [ReaShure, Balchem Corp., New Hampton, NY]). Diets were formulated according to nutrient requirements of dairy cattle (NRC, 2001) to meet the nutrient requirements of the cows (milk yield= 45 kg/d, milk fat= 3.3% and milk true protein= 2.9%). Offered ad libitum diets (TMR, at 0730, 1530 and 2330 h) aimed to have refusals of 5 to 10%. The rumen protected Met, Lys and Chol were fed top-dressed to the LPD receiving cows. All the cows were milked three times a day (0630, 1430 and 2230 h).
2.3. Statistical Analysis
Obtained data for DMI, milk yield and milk composition were analyzed using PROC MIXED of SAS (SAS software version 9.4) as repeated measurement. The statistical model was:
Yijk = μ + Ci + Tj + CTij+ Pk + TPjk + eijk
where Yijk is the dependent variable, μ is the overall mean, Ci is the cow, Tj is the experimental diet (jth treatment), CTij is the interaction of cow × treatment, Pk is the experimental phase (phase 1 & 2), and TPjk is the interaction of treatment × phase and eijk is the error term.
Collected data for blood parameters and reproductive performance were analyzed using PROC GLM of SAS (SAS software version 9.4). The statistical model was:
Yij = μ + Ci + Tj + Pk + PTjk + eij
where Yijk is the dependent variable, μ is the overall mean, Ci is the cow, Tj is the jth treatment ( experimental diet), Pk is the experimental phase (phase 1 & 2), and TPjk is the treatment × phase interaction, with the error term eijk assumed to be normally distributed. P-value of 5% (P≤ 0.05) were declared as significant difference among treatments.
Results and discussion: Daily DMI, milk protein and fat were not significantly different, but significantly higher milk yield were achieved for the control, LPDML and LPDMLC groups (P<0.05). Significantly higher milk urea N (13.82 mg/dl) was obtained for the control group (P<0.05). Plasma concentration of insuline and creatinine was not affected by experimental diets, whereas significantly higher concentration of glucose (58.75 mg/dl) was obtained for LPDL (P<0.05). Energy status indicators (NEFA&BHBA) were not influenced by experimental diets, but blood urea-N was significantly higher (16.15 mg/dl) for the control group (P<0.05). Number of days from calving to first artificial insemination, open days, number of artificial inseminations, number of first services resulting in pregnancies and number of second services resulting in pregnancy were not significantly different among the experimental groups. In conclusion, the results showed that it might be possible to keep dairy cattle productive and reproductive performance by decreasing about 12.35% of the CP level of the diet which was compensated for by supplementing RPMet, RPLys and RPChol.