Effect of Dietary Fat on the Metabolism of Energy and Nitrogen, Serum Parameters, Rumen Fermentation, and Microbiota in twin Hu Male Lambs

Background Fat is the main substance that provides energy to animals. However, the use of fat in twin Hu lambs has not been investigated. Thirty pairs of male twin lambs were examined to investigate the effects of dietary fat on the metabolism of energy and nitrogen, ruminal fermentation, and microbial communities. The twins are randomly allotted to two groups (high fat: HF, normal fat: NF). Two diets of equal protein and different fat levels. The metabolism test was made at 50-60 days of age. Nine pairs of twin lambs are slaughtered randomly, and the rumen fluid is collected at 60 days of age. Results The initial body weight (BW) in the HF group did not differ from that of NF group (P > 0.05), but the final BW was tended to higher than that of NF group (0.05 < P < 0.1). The digestive energy (DE), metabolism energy (ME), DE/ME in the HF group tend to be higher than those in the NF group (0.05 < P < 0.1). Ruminal ammonia nitrogen (NH3-N) and the proportion of total volatile fatty acids (TVFA) are higher than that in the NF group (P < 0.05). A high throughput sequencing analysis reveals that there were no differences between the two groups in terms of the richness estimates and diversity indices (P > 0.05). The Proteobacteria and Fibrobacteres phyla were higher than that in NF group (P<0.05). Conclusions This study demonstrated that high fat diet before weaning can affect the abundance of several groups of rumen bacteria in rumen, such as significantly increasing phyla Proteobacteria and Fibrobacteres, and genera of Succinivibrio, Alloprevotella, and Saccharofermentans, but significantly decreasing genera of Clostridium IV, Dialister, Roseburia, and Butyrivibrio. And high fat diet improved the performance of lambs at weight gain, energy utilization, and had effect on VFA composition but no effects on serum enzymes and serum hormone.


Introduction
In human, infant formula is designed to meet all the nutritional needs to promote 39 infant growth and development. Early nutrition has critical effects on the long-term 40 health of adult animals [1,2]. Therefore, it is important to understand the early 41 nutritional regulation of young animals. It is reported that calves fed on an elevated heifers to reach breeding size earlier with lower production costs. Other researchers 48 suggested that fat may be the result of the dietary fat, fat type, additive amounts and 49 interactions. 50 The influence of dietary fat vary among studies, which could be associated with 51 species of animals, type and concentrations of fat and dietary composition [8,9].   In this study, 30 pairs of twin Hu male lambs at seven days old (BW = 4.22±0.56 76 kg) were obtained randomly and assigned to two groups within block to 20 pens (pen=3 77 animals, 10 pens/group). All the lambs were weighed and ear-tagged before the start of 78 the experiment, then they were subjected to normal immunisation procedures. Two 79 diets of equal protein but different fat levels were fed to the lambs until 60 days of age: 80 one was a normal-fat diet consisting of a milk replacer (MR; 15% fat, which followed 81 industry standard 'milk replacer for lamb NY/T2999-2016' in China and patent 'a milk 82 replacer for calf and lamb ZL02128844.5') and a starter (2.8% fat), and the other was 83 a high-fat diet consisting of MR (27% fat) and a starter (5.07% fat). Table 1 presents 84 the chemical constituents and ingredients of the trial diets. All the lambs were fed MR 85 at 2% of BW from 7 to 50 days of age and 1.5% of BW from 50 to 60 days of age. One-86 third of the MR was fed at 06:00, one-third was fed at 12:00 and the remainder were 87 fed at 17:30. From day 50 to 60, the lambs were fed twice daily at 6:00 and 17:30, 88 allowing 5-10% orts, and fresh drinking water and starter were provided ad libitum.  at P < 0.05, and 0.05 < P < 0.10 was designated as a tendency. Alpha diversity indices 169 (Chao, Simpson, Shannon) were generated with the QIIME pipeline, whereas diversity 170 (i.e., diversity between groups of samples) was used to create principal coordinate 171 analysis (PCoA) plots using unweighted distances. The community structure analysis 172 histograms were produced according to taxonomy using greengenes database.

Digestion and Metabolism of Energy and Nitrogen
175 Table 2 lists the results of BW, energy and nitrogen digestion and metabolism.

176
The initial BW in the HF group did not differ from that of NF group (P > 0.05), but the 10 177 final BW was tended to higher than that of NF group (0.05 < P < 0.1). The milk replacer 178 intake in the HF group was higher than that of NF group (P < 0.05), however, the starter 179 intake was tend to lower than that of NF group (0.05 < P < 0.1), the feed conversion 180 rate had no differ from that of NF group (P > 0.05). The GE of feed intake, faecal energy 181 (FE) and urine energy (UE) in the HF group did not differ from that of the other group 182 (P > 0.05). The DE, ME, and DE/ME were higher in the NF group (0.05 < P < 0.1).

183
Lambs fed HF diets had no differ apparent digestibility of GE and ME/DE compared 184 to the NF group (P > 0.05). The intake N, faecal N, urine N, retained N, absorbed N, 185 and biological values did not differ between the two groups (P > 0.05); however, the 186 utilisation of N was higher than the NF group (0.05 < P < 0.1).   between the two groups in terms of the richness estimates and diversity indices (P > 223 0.05) (Fig. 1, Table 5).  Next, we analysed the beta diversity. Fig. 2 presents the principal coordinate in the HF group. These results confirmed that the differences between these groups 237 were greater than those within the groups, but there was no difference between the two 238 groups in terms of the microbial communities.  Fibrobacteres phyla were higher than that in NF group (P<0.05). Other phyla were no 254 significant differences (P > 0.05).

295
Six taxa displayed significant differences in their abundance levels between the 296 NF and HF groups, using LDA score log10 > 2.0 (Fig. 5A). The liner discriminant  the HF group, which shows none of these potential functional (Fig. 6). More

358
That may be the fat in HF diet concentration in the range of animal autoregulation.

359
Normal pH values range from 6.4-6.8 [28]. The ruminal pH in each group in the present 360 study appears to be within the normal range (6.71-6.77), which indicates that the 361 internal environment of the rumen is relatively static when lambs are given the HF diet. fermentation [38,39]. Jami and Mizrahi [40] reported that the proportion of