Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on -12, 10, 24, and 136 DIM for gene expression analysis. Growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear factor 4alpha (HNF4alpha), fibroblast growth factor-21 (FGF-21), and peroxisome proliferator-activated receptor alpha (PPARalpha) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41 x 10(4) message copies for -12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4alpha mRNA was not altered by DIM or any of the treatments. Abundance of PPARalpha mRNA was unchanged through 24 DIM but increased by 136 DIM. There was a trend for an interaction of bST, diet, and DIM on PPARalpha mRNA abundance from 24 to 136 DIM because the amount of PPARalpha mRNA increased in SSN, SSY, and AFN cows but was not altered in AFY cows. The amount of FGF-21 mRNA increased markedly in early lactation but, like HNF4alpha mRNA, was not affected by bST, diet, or their interactions. These results indicate 1) that bST induced increases in hepatic expression of GHR, IGF-I, and IGFBP3 when cows were in negative energy balance in early lactation, 2) there was no effect of reduced dietary n-6/n-3 content on hepatic gene expression, and 3) there was support for a potential homeorhetic role of hepatic FGF-21 via uncoupling the somatotropin-IGF-axis in early lactation.

Nutrition and suckling are largely recognised as the most important factors affecting the postpartum period and consequently the reproductive efficiency of beef cattle. The aim of this study was to evaluate the effects of body condition score (BCS) and suckling restriction with and without the presence of the calf on milk production, reproductive efficiency and calf performance. Sixty-three crossbred (Angus · Hereford) multiparous cows were managed to maintain different BCS at calving and thereafter (low vs moderate; L, n = 31 and M, n = 32). Within each group of BCS (L and M) at week 9 postpartum (66 AE 0.88 days postpartum) cows were assigned to three suckling treatments (ST): (i) suckling ad libitum (S, n = 20); (ii) calves fitted with nose plates during 14 days remaining with their dams (NP, n = 22); and (iii) calves were completely removed from their dams for 14 days, and thereafter returned (CR, n = 21). Milk production was assessed by milking procedure at Day 65 (the day before onset of ST) and every 20-22 days until the end of the experiment. Cows were bled via jugular venipuncture every 28 days from Day -98 (Day 0 = calving) until Day 66. From Day 66 cows were bled every 7 days until the end of the mating period (Day 128). Concentrations of progesterone, non-esterified fatty acids and b-hydroxybutyrate acid and insulin were measured. Presence of corpus luteum (CL) was recorded and maximum follicle diameter was measured in all cows from the onset of the ST (Day 66) and during the following 4 weeks (until Day 94) in a weekly frequency. At Day 94, more cows (P < 0.001) in NP and in CR had CL compared with S cows (68, 57 and 21% for NP, CR and S, respectively). At that time, more cows in M-BCS presented CL than cows in L-BCS (77 vs 25; P < 0.0001). Within M-BCS, there were no differences in milk production between ST groups, while L-BCS cows with NP or CR produced less milk than S cows. Calf liveweight at weaning was 159.3 AE 3.1, 150.1 AE 2.9 and 147.0 AE 3.1 kg for S, NP and CR, respectively (P < 0.001). Suckling restriction with and without the presence of the calf had similar effects on reproductive performance, milk production and calf growth, while BCS interacted with ST to influence milk production. These results indicate that temporary suckling restriction could be an excellent management tool to increase reproductive performance of cows in moderate condition.

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The objective of this study was to investigate the effect of different body condition score (BCS) at 30 days before calving (230 days) induced by a differential nutritional management from 2100 days until 230 days on productive parameters, the interval to first ovulation and blood parameters in primiparous and multiparous Holstein cows under grazing conditions until 60 days post partum. The experimental arrangement was a randomized complete block design, where cows were blocked according to BW and expected calving date and then randomly assigned to different nutritional treatments from -100 to -30 days relative to calving to induce different BCS. As the assignment of cows to treatments was random, cows had to lose, maintain or gain BCS; thus, different planes of nutrition were offered with approximately 7, 14 or 20 kg dry matter per day. The BCS score was assessed every 15 days and animals were reassigned in order to achieve the desired BCS at -30 days. Only animals that responded to nutritional treatment were considered and this was defined as follows: primiparous and multiparous high cows (PH and MH) had to gain 0.5 points of BCS, primiparous low (PL) had to lose 0.5 points of BCS and multiparous low (ML) had to maintain BCS at least in two subsequent observations from 2100 to 230 days. From 230 days to calving, primiparous and multiparous cows (P and M cows) were managed separately and cows were offered a diet once a day. From calving to 60 days post partum, cows of different groups grazed in separate plots a second year pasture. Cows were also supplemented individually with whole-plant maize silage and commercial concentrate. Cows had similar BCS at 2100 days and differed after the nutritional treatment; however, all groups presented similar BCS at 21 days post partum. The daily milk production and milk yield at 60 days post partum was higher in M than P cows. The percentage of milk fat was higher in PH cows compared with PL cows. Concentrations of non-esterified fatty acids (NEFA) were affected by the BCS at 230 days within parity, and in PH cows the concentration of NEFA was higher than in PL cows. The concentrations of total protein were higher in M cows. A lower probability of cycling was found in PL than in PH cows (P , 0.05) and in ML than in MH cows (P , 0.05). Treatment affected various endocrine/metabolic profiles according to parity, suggesting that the metabolic reserves signal the productive/reproductive axis so as to induce a differential nutrient partitioning in adult v. first-calving cows.

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The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Early lactation is an energy-demanding period for dairy cows which may lead to negative energy balance, threatening animal health and consequently productivity. Herein we studied hepatic mitochondrial function in Holstein-Friesian multiparous dairy cows during lactation, under two different feeding strategies. During the first 180 days postpartum the cows were fed a total mixed ration (70% forage: 30% concentrate) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Mendicago sativa plus supplementation (grazing group, G1). From 180 to 250 days postpartum, all cows grazed Festuca arundinacea and were supplemented with total mixed ration. Mitochondrial function was assessed measuring oxygen consumption rate in liver biopsies and revealed that maximum respiratory rate decreased significantly in grazing cows during early lactation, yet was unchanged in non-grazing cows during the lactation curve. While no differences could be found in mitochondrial content or oxidative stress markers, a significant increase in protein lysine acetylation was found in grazing cows during early lactation but not in cows from the non-grazing group. Mitochondrial acetylation positively correlated with liver triglycerides and β-hydroxybutyrate plasma levels, well-known markers of negative energy balance, while a negative correlation was found with the maximum respiratory rate and sirtuin 3 levels. To our knowledge this is the first report of mitochondrial function in liver biopsies of dairy cows during lactation. On the whole our results indicate that mitochondrial function is impaired during early lactation in grazing cows and that acetylation may account for changes in mitochondrial function in this period. Additionally, our results suggest that feeding total mixed ration during early lactation may be an efficient protective strategy.

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