## 4 Discussion
### In Vitro Bihydrogenation and Fatty Acid Concentrations  
The present study demonstrated the advantageous reducing effects of SAG, PIN, and CLO at specific dose levels on the in vitro concentrations of C14:0 and C16:0. The saturated fatty acids, particularly C14:0 and C16:0, have been extensively studied and are known to possess atherogenic and thrombogenic properties, as documented by [PERSON] et al. [60]. Notably, various studies, including those by [PERSON] et al. [61] and [PERSON] et al. [62], have consistently reported their ability to elevate cholesterol levels and promote platelet aggregation. The consumption of atherogenic and thrombogenic foods, which exhibit a propensity to induce platelet aggregation, is associated with an increased risk of cardiovascular disease and, consequently, offers limited nutritional benefits. During the ruminal fermentation process, LA undergoes an initial isomerization step, resulting in the formation of _cis_-9, _trans_-11 CLA. Subsequently, CLA is converted to _trans_-11 C18:1 and further to C18:0 [63]. In our study, the addition of 300 mg/L and 600 mg/L of CLO resulted in a significant reduction in the bihydrogenation of LA and LNA. Interestingly, we observed an accumulation of _cis_-9, _trans_-11 CLA and _trans_-11 C18:1, along with decreased concentrations of C18:0 in these treatments, indicating partial bihydrogenation of LA and LNA in the presence of CLO at both inclusion levels. This accumulation of CLA and _trans_-11 C18:1, coupled with the decreased concentration of C18:0, suggests that the second step of bihydrogenation is inhibited by CLO at 300 mg/L and 600 mg/L. Several studies have previously reported the hydrogenation of C18:1 isomers to C18:0 or their isomerization into various positional and geometric isomers [64; 65]. The inhibitory effects of herbal essential oils on the growth of microorganisms involved in the biohydrogenation of _trans_-11 C18:1 to C18:0 have been reported [66; 67]. This observation could potentially elucidate the decreased levels of C18:1 _n_-9 observed in several treatments examined in our study. Additionally, the concurrent reduction of C18:0 alongside the elevation of _trans_-11 C18:1 concentrations suggests that the essential oils primarily facilitated the conversion of C18:1 _n_-9 to _trans_-11 C18:1. The available evidence strongly indicates that the elevation of _cis_-9, _trans_-11 CLA levels in ruminant tissues and milk predominantly originates from an augmented availability of _trans_-11 C18:1 [68]. Several studies have investigated the efficacy of marine lipid supplements (fish oil and microalgae products) as potential sources of 20:5 _n_-3 and 22:6 _n_-3 for dairy ruminants [67; 69; 70]. However, the transfer rates of these very long chain PUFAs into meat and milk are constrained due to the extensive biohydrogenation in the rumen. In the context of ruminal metabolism, the conversion of C20:5 _n_-3 and C22:6 _n_-3 is thought to undergo a preliminary isomerization process, similar to the behavior observed for the C18 PUFAs. This isomerization step is believed to give rise to conjugated intermediates consisting of 20 and 22 carbon atoms [71]. In the current study, the level of biohydrogenation of C20:5 _n_-3 and C22:6 _n_-3 was comparatively lower in contrast to the ruminal bihydrogenation of C18 fatty acids such as C18:2 _n_-6 and C18:3 _n_-3. In addition, it is important to acknowledge that a complete understanding of the biohydrogenation pathways of C20:5 _n_-3 and C22:6 _n_-3 is still lacking. Our results align with those of [PERSON] et al. [72], who demonstrated a biohydrogenation rate of 61.6% for EPA and 46.2% for DHA following 24 h of incubation when a 2% fish oil inclusion was incorporated into the diets. According to compelling evidence, both general dietary recommendations and guidelines from the World Health Organization (WHO) propose that replacing SFA with PUFA can effectively lower the risk of cardiovascular disease (CVD) by reducing LDL-cholesterol levels [73; 74].