Piscopo, Federica (2025) Development of a corrective strategy for the negative energy balance phase in high profitability dairy cows. [Tesi di dottorato]

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Abstract

This thesis aims to explore the reproductive and metabolic challenges faced by high-producing dairy animals during the period around calving. High-producing dairy cows have a pivotal role in the global dairy farm economy, satisfying the increasing milk demand worldwide. However, high-producing dairy cows often experience an increased incidence of reproductive inefficiency, most of which is caused by impaired postpartum. Indeed, the postpartum period is often associated with a high incidence of most dairy cattle diseases and a high risk of removal from the herd. Moreover, the occurrence of a period of Negative Energy Balance (NEB) around calving together with an increased risk of inflammatory diseases has been observed. During NEB, the energy requirements to support lactation exceed the energy intake, which leads to the mobilization of body fat reserves resulting in an increase of Non-Esterified Fatty Acids (NEFAs) in the blood and the follicular fluid. In the latter, this condition can result in excessive saturated fatty acids (SFAs) levels, compromising the oocyte developmental competence. Escherichia Coli is one of the first pathogens that reaches the reproductive tract during uterine diseases after calving and can induce an inflammatory response due to the presence of a cell wall component, an endotoxin, the lipopolysaccharide (LPS) in the blood and follicular fluid. Granulosa cells initiate an inflammatory response, in response to the presence of the LPS binding the toll-like receptor 4 (TLR4), which increases the production of pro-inflammatory cytokines. The LPS presence in follicular fluid is detrimental to the oocyte and thus compromises overall fertility. This thesis focuses on the potential protective role that Oleic Acid (OA) can have against high levels of SFAs and LPS. The OA is a mono-unsaturated fatty acid (MUFA) known for its anti-lipotoxic and anti-inflammatory effects. To this end, this research has been divided into two main parts: an in vitro and an in vivo study. In the in vitro study, the OA's role in counteracting the toxic effects of high SFA levels and LPS during the in vitro maturation of bovine oocytes has been tested. In particular, the study was conducted with four progressive experiments. In the first experiment, the effect of 250 µM OA towards 10 µL/mL LPS in a serum-free maturation media was assessed on cleavage rate and blastocysts rate. Unexpectedly, no differences were found among the groups. In the second experiment, based on the results obtained, the effect of 10 µL/mL LPS in a serum-free or serum-supplemented maturation media was assessed on cleavage rate and blastocysts rate. The results confirmed the detrimental effect of LPS in a serum-supplemented media (P<0.05). In the third experiment, the effect of 250 µM OA towards 10 µL/mL LPS in a serum-supplemented maturation media was assessed on cleavage rate and blastocysts rate. The LPS significantly (P<0.05) impacted both cleavage and blastocyst rates, which were completely restored by the addition of OA (P<0.05). In the fourth experiment, the protective role of 200 µM OA against the combination of SFAs (100 µM Stearic Acid, and 150 µM Palmitic Acid) with 10 µL/mL LPS was assessed on cleavage and blastocyst rates and on mRNA expression of several genes related to apoptosis (BAX), cumulus expansion (AREG and EREG) and inflammation (IL-8, TNFα, and TLR4) detected in cumulus cells and oocytes. Unexpectedly, the main detrimental effect was given by the presence of SA and PA (P<0.05), with no additional effect given by the LPS. The presence of the OA completely restored cleavage and blastocyst rates (P<0.05). The relative gene expression of BAX, AREG, EREG, IL-8, TNFα, and TLR4 was not different among groups both for oocytes and cumulus cells, except for the EREG relative expression in cumulus cells in the group of SA+PA+OA that was significantly upregulated (P<0.05) compared to the control and LPS and SA+PA. Based on these results, in the in vivo study, an OA-supplemented diet was formulated to see if OA could mitigate the metabolic stress associated with the NEB period. To this aim, eight-month pregnant Holstein Friesian heifers were randomly divided into two groups (5 vs 6) to receive a standard, palmitic acid-rich, or rumen-protected oleic acid-rich diet from 4 weeks before calving until 4 weeks post-calving. NEFA, β-Hydroxybutyric acid, and haptoglobin profiles in blood were monitored, and cumulus-oocyte-complexes (COCs) were collected via transvaginal ovum pick-up (OPU) at 8, 12, and 16 weeks postpartum for in vitro maturation, fertilization, and culture until day 8. Oleic acid supplementation resulted in higher levels (P<0.05) of OA in blood and reduced (P<0.05) PA, furthermore, 1.6 more oocytes were collected with no differences in developmental competence. These findings suggested that peripartum oleic acid supplementation can support follicles and oocytes during NEB development. The overall conclusion is a confirmed beneficial role of the OA; however, further studies are needed to better investigate the potential long-term effects of OA supplementation on fertility in dairy cows during the transition period.

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