# Effects of dietary supplementation with linseed and Padina pavonica algae extract on semen quality and tissue fatty acid metabolism in rabbit bucks

Francesca Di Federico, Simona Mattioli, Elisa Angelucci, Gabriele Brecchia, Nour Fehri, Alda Quattrone, Davi Savietto, Bairem Jemmali, Yasmein Z Abdel-Ghafar, Sebastiana Failla, et al.

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Francesca Di Federico, Simona Mattioli, Elisa Angelucci, Gabriele Brecchia, Nour Fehri, et al.

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ijas

Italian Journal of Animal Science
ISSN 1828-051X

www.italianjournalofanimalscience.com
Volume 21
January 2023

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ISSN: 1828-051X (Online) Journal homepage: www.tandfonline.com/journals/tjas20

# Effects of dietary supplementation with linseed and *Padina pavonica* algae extract on semen quality and tissue fatty acid metabolism in rabbit bucks

Francesca Di Federico, Simona Mattioli, Elisa Angelucci, Gabriele Brecchia, Nour Fehri, Alda Quattrone, Davi Savietto, Bairem Jemmali, Yasmein Z. Abdel-Ghafar, Sebastiana Failla, Olimpia Barbato, Michel Colin, Mathieu Guillevic, Cesare Castellini & Alessandro Dal Bosco

**To cite this article:** Francesca Di Federico, Simona Mattioli, Elisa Angelucci, Gabriele Brecchia, Nour Fehri, Alda Quattrone, Davi Savietto, Bairem Jemmali, Yasmein Z. Abdel-Ghafar, Sebastiana Failla, Olimpia Barbato, Michel Colin, Mathieu Guillevic, Cesare Castellini & Alessandro Dal Bosco (2025) Effects of dietary supplementation with linseed and *Padina pavonica* algae extract on semen quality and tissue fatty acid metabolism in rabbit bucks, Italian Journal of Animal Science, 24:1, 1609-1620, DOI: 10.1080/1828051X.2025.2540031

**To link to this article:** https://doi.org/10.1080/1828051X.2025.2540031

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# Effects of dietary supplementation with linseed and *Padina pavonica* algae extract on semen quality and tissue fatty acid metabolism in rabbit bucks

Francesca Di Federico<sup>a</sup>, Simona Mattioli<sup>a</sup>, Elisa Angelucci<sup>a</sup>, Gabriele Brecchia<sup>b</sup>, Nour Fehri<sup>b</sup>, Alda Quattrone<sup>b</sup>, Davi Savietto<sup>c</sup>, Bairem Jemmali<sup>d</sup>, Yasmein Z. Abdel-Ghafar<sup>e</sup>, Sebastiana Failla<sup>f</sup>, Olimpia Barbato<sup>g</sup>, Michel Colin<sup>h</sup>, Mathieu Guillevic<sup>i</sup>, Cesare Castellini<sup>a</sup> and Alessandro Dal Bosco<sup>a</sup>

## ABSTRACT

This study aimed to evaluate the effects of dietary supplementation with 5% extruded linseed, with or without 0.2% *Padina pavonica* algae extract, on semen quality and fatty acid metabolism in rabbit bucks. Forty-five New Zealand White rabbit bucks were divided into three groups (15 per diet) and fed for 120 days either a control diet (C), a diet supplemented with 5% linseed (L), or a linseed diet plus 0.2% algae extract (LPP). The semen characteristics, the fatty acid composition of blood, liver, and testis, and estimated indexes of lipid metabolism were assessed. The results showed that the use of linseed in both L and LPP diets had a significant effect on some semen traits (concentration, progressive motility, linearity, amplitude of lateral head displacement, beat cross frequency, acrosome reacted/capacitated sperm) compared to C group. In the tissues analysed, significant effects were observed only in relation to the linseed administration. In conclusion, while linseed positively influenced sperm quality and lipid composition of tissues, the *Padina pavonica* extract had only a minor effect, limited to hepatic DHA level.

## HIGHLIGHTS

* The *Padina pavonica* algae as enhancer of n-3 PUFA metabolism was tested.

* Dietary linseed administration improved progressive motility and linearity of rabbit sperm.

* The *Padina pavonica* algae positively modulated the lipid metabolism of rabbit liver.

## ARTICLE HISTORY

Received 9 June 2025

Revised 16 July 2025

Accepted 22 July 2025

## KEYWORDS

Rabbit; linseed; *Padina pavonica* algae; semen; polyunsaturated fatty acids

## Introduction

Rabbit semen is a heterogeneous matrix composed by spermatozoa (SPZ), seminal plasma (SP), prostatic secretory granules (PSG) along with many other compounds. In detail, the membrane of SPZ, which represents the main component of semen, is mainly composed by polyunsaturated fatty acids (PUFA) and cholesterol (Castellini et al. 2006). The long chain PUFA (C ≥ 20 C; LC-PUFA) present in the SPZ membrane of rabbits belong to the omega-6 (n-6) series, mainly docosapentaenoic acid (C22:5n-6, DPA; Poulos et al. 1973; Darin-Bennett et al. 1974; Mourvaki et al. 2010), whereas in other mammalian species, the docosahexaenoic acid (C22:6n-3, DHA; Neill and Masters

1972; Lin et al. 1993) represents the majority of the LC-PUFA. However, many studies demonstrated that unbalanced diets in terms of omega-3 (n-3) and n-6 fatty acids (mainly constituted by linoleic acid, C18:2n-6, LA) modify the fatty acid profile of rabbit spermatozoa and of reproductive organs (Mattioli et al. 2023), producing an excess of arachidonic acid (C20:4n-6, AA).

Since mammals, unlike lichens and soil algae (Burdge 2018), cannot synthesise PUFA, dietary precursors are necessary. The LA and the alpha-linolenic acid (C18:3n-3, ALA) are essential fatty acids, being the precursors of the corresponding LC-PUFA. They come from vegetable products and are converted by the

<footnote><sup>a</sup>Dipartimento di Scienze Agrarie, Ambientali e Alimentari, Università di Perugia, Perugia, Italia; <sup>b</sup>Dipartimento di Medicina Veterinaria e Scienze Animali, Università di Milano, Lodi, Italia; <sup>c</sup>GenPhySE, INRAE, Université de Toulouse, ENVT, Castanet-Tolosan, Francia;</footnote>

<footnote><sup>d</sup>University of Carthage, Mateur, Higher School of Agriculture, Tunisi, Tunisia; <sup>e</sup>Agriculture Research Center (ARC), APRI, Animal Production Research Institute (APRI), Dokki, Giza, Egypt; <sup>f</sup>Centro Ricerche Produzioni Animali e Acquacoltura, CREA, Monterotondo, Roma, Italia; <sup>g</sup>Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia, Perugia, Italia; <sup>h</sup>COPRI Sarl, Ploudalmezeau, France; <sup>i</sup>Valorex, la Messayais, Combourtillé, France</footnote>
liver (Cook, 1996), and in a lesser extent by the testicle (Castellini et al. 2022) specifically by Sertoli's cells (Retterstøl et al. 1998), or by ovaries (Mattioli et al. 2021a) into LC-PUFA thanks to specific enzymes: desaturases (Δ5- and Δ6-desaturase) and elongases (2- and 5-elongase).

The role of PUFA, especially of the n-3 series, on human/animal health is well known; in particular, eicosapentaenoic acid (C20:5n-3, EPA) and DHA, positively modulate many physiological processes, including the regulation of lipid levels in plasma and tissues (Li et al. 2021), cardiovascular (Cottin et al. 2011) and immune function (Wu and Meydani 1998), glucose metabolism (Shimura et al. 1997), neuronal development (Cao et al. 2009), visual activity (Mitchell et al. 2003), and male and female fertility (Hosseini et al. 2019). In this regard, it should be noted how dietary supplementation (Calder 2002; Givens 2009) modify the lipid structure of cells, such as an enrichment of DHA that is a constituent of the phospholipid membrane of the retina, brain, reproductive tissues, and gametes, or EPA, eicosanoid precursors of prostaglandins and thromboxanes, as well as the hormone synthesis (Kujoana et al. 2024).

The PUFA are also fundamental to improve the fluidity and movement of spermatozoa (Castellini et al. 2019) being involved in a whole series of processes, such as the capacitation and acrosomal reaction, essential for oocyte fertilisation (Flesch and Gadella 2000).

Many studies identified the benefits of incorporating extruded linseed as a source of ALA (Castellini et al. 2004; Curone et al. 2022) on the semen quality of rabbits. Castellini et al. (2019) reported that 10% dietary supplementation of linseed, opportunely protected with an antioxidant (200 mg/kg of alpha-tocopheryl acetate), and administered for 110 days improved the fatty acid profile and the semen quality of rabbit bucks. However, a supplementation of 10% of linseed is expensive, as well as causing technical problems in the preparation of diets, given its high fat content.

The present research evaluates the effect of linseed and/or *Padina pavonica* algae (a small brown alga found in the Indian Ocean, the Pacific Ocean, the Atlantic Ocean and the Mediterranean Sea) incorporation on semen quality of rabbit bucks and in the activation of lipid metabolism as well as a large amount of nutraceutical effects (Pangestuti et al. 2022).

Other strategies explored the effect of different sources of PUFA on reproductive traits of several animal species and humans (Wathes et al. 2013; Van Tran

et al. 2017; Maranesi et al. 2018; Rodríguez et al. 2019). In particular, extensive studies highlight a particular role of some brown algae, such as *Padina pavonica*.

It should be noted that reproductive studies in rabbit are easily transferable to human (Castellini et al. 2024). Furthermore, in rabbit, semen collection is very easy and does not require the killing of the animal (Lucio et al. 2009), and dietary plan, unlike humans, are easily standardised and reproducible (Theau-Clément et al. 1998).

Therefore, since PUFA are important elements of semen, and linseed contains high levels of ALA, while the *Padina pavonica* is considered a stimulator of lipid metabolism, the aim of this study was to verify the dietary effect of low amount of PUFA n-3 (5% extruded linseed) combined or not with *Padina pavonica* algae extract (0.2%) in the interest of minimising linseed use.

# Materials and methods

## Ethics

All methods are reported following ARRIVE guidelines for the reporting of animal experiments. This study was carried out in accordance with the Guiding Principles in the Use of Animals for Scientific Purposes. It was approved by the Animal Ethics Monitoring Committee of the University of Perugia (CEL AOUS; authorisation n° 325/2021-PR; protocol 2B818.83).

## Animals and diets

Forty-five New Zealand White rabbit bucks (6 months of age) were reared in a commercial rabbitry (Brachino Patrizia, Bagnoregio, VT, Italy) in multilevel platform cages (1.0 W x 1.83 L x 1.0H m) and randomly divided into three experimental groups, 15 rabbits for each group. The experimental groups were subsequently checked to ensure comparable characteristics regarding body weight, seminal traits, and other related variables. Rabbits were fed three different diets (Table 1):

* Control (C), commercial standard diet;

* Linseed group (L), C diet enriched with 5% of extruded linseed;

* Linseed and algae group (LPP), L diet plus *Padina Pavonica* algae at 0.2% (the dose recommended by the manufacturer for rabbit).

Throughout the experimental period (from 24 to 48 weeks of life) the room temperature ranged from
<figure><img src="image_16.png" /><figcaption>**Table 1.** Formulation and calculated composition (%, UI, kcal) of diets.</figcaption></figure>

<table><thead><tr><th>Ingredients</th><th>C</th><th>L</th><th>LPP</th></tr></thead><tbody><tr><td>Wheat bran</td><td>25.07</td><td>24.87</td><td>24.90</td></tr><tr><td>Barley</td><td>13.33</td><td>13.00</td><td>13.00</td></tr><tr><td>Sunflower seed</td><td>12</td><td>11.67</td><td>11.50</td></tr><tr><td>Alfalfa meal</td><td>10.83</td><td>13.00</td><td>13.00</td></tr><tr><td>Sunflower husks</td><td>10.00</td><td>10.00</td><td>10.00</td></tr><tr><td>Beet pulp</td><td>7.50</td><td>5.66</td><td>5.50</td></tr><tr><td>Extruded linseed</td><td>—</td><td>5.00</td><td>5.00</td></tr><tr><td>Full-fat soybean</td><td>5.00</td><td>2.95</td><td>3.10</td></tr><tr><td>Wheat straw</td><td>4.17</td><td>2.00</td><td>2.00</td></tr><tr><td>Molasses cane</td><td>3.00</td><td>3.00</td><td>3.00</td></tr><tr><td>Wheat</td><td>2.50</td><td>2.50</td><td>2.50</td></tr><tr><td>Grape seed meal</td><td>2.33</td><td>1.67</td><td>1.70</td></tr><tr><td>Soya hulls</td><td>0.00</td><td>0.16</td><td>1.70</td></tr><tr><td>Calcium carbonate</td><td>1.60</td><td>1.48</td><td>1.42</td></tr><tr><td>Soybean oil</td><td>0.78</td><td>—</td><td>—</td></tr><tr><td>Sodium chloride</td><td>0.40</td><td>0.40</td><td>0.4</td></tr><tr><td>palm oil</td><td>0.33</td><td>—</td><td>—</td></tr><tr><td>Carboxymethylcellulose</td><td>0.30</td><td>0.30</td><td>0.3</td></tr><tr><td>Oligo vitamin supplement<sup>a</sup></td><td>0.25</td><td>0.25</td><td>0.25</td></tr><tr><td>Padina Pavonica algae</td><td>—</td><td>—</td><td>0.2</td></tr><tr><td>Lysine HCL</td><td>0.16</td><td>0.17</td><td>0.17</td></tr><tr><td>Liquid Acidifer</td><td>0.15</td><td>0.15</td><td>0.15</td></tr><tr><td>Magnesium oxide</td><td>0.10</td><td>0.1</td><td>0.1</td></tr><tr><td>Methionine hydroxyanalog</td><td>0.06</td><td>0.07</td><td>0.07</td></tr><tr><td>Liquid choline</td><td>0.05</td><td>0.05</td><td>0.05</td></tr><tr><td>Vitamine E</td><td>0.03</td><td>0.03</td><td>0.03</td></tr><tr><td>L Threonine</td><td>0.03</td><td>0.01</td><td>0.01</td></tr><tr><td>DL Methionine</td><td>0.03</td><td>—</td><td>—</td></tr><tr><td><em>Chemical composition</em></td><td></td><td></td><td></td></tr><tr><td>Crude Protein</td><td>16.5</td><td>16.6</td><td>16.5</td></tr><tr><td>Lipids</td><td>3.62</td><td>3.91</td><td>3.96</td></tr><tr><td>Fiber</td><td>17.16</td><td>16.82</td><td>16.91</td></tr><tr><td>Ash</td><td>7.99</td><td>8.00</td><td>8.09</td></tr><tr><td>DE (kcal/Kg)<sup>b</sup></td><td>2350</td><td>2350</td><td>2350</td></tr></tbody></table>

C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed + 0.2% *Padina Pavonica* algae; ADF: acid detergent fiber; CP: crude protein; DE: Digestible Energy; EE: ether extract.

<sup>a</sup>Vitamin mineral premix composition: Vitamin A 6,000,000 IU, D3 600,000 IU, E 20,000 IU, K3 1200 mg, B1 800 mg, B2 1600 mg, B6 800 mg, B12 6.0 mg, Biotin 60.0 mg, Niacinamide 16,000 mg, Folic acid 400 mg, Calcium pantothenate 6666 mg. 2 Liquid acidifier composition: Formic acid 75%.

<sup>b</sup>DE: estimated with the following equation: 14.2 – 0.205 ADF + 0.218 EE + 0.057 CP (Fernández-Carmona et al. 1996).

+15 to +20°C and the relative humidity from 65 to 70%.

The diets were formulated by Martini s.r.l (Longiano, FC, Italy) to be isoenergetic and isoproteic and were administered for a total of 120 days. The L and LPP diets had a supplement of 0.03% of vitamin E to control lipid oxidation.

The total experimental trial lasted 180 days; 60 days of training, 60 days of adaptation to the diets, and 60 days of data collection when blood and semen samples were collected. During the training period, the *libido* (i.e. seconds between the introduction of a female rabbit and male ejaculation) and semen traits (volume, concentration, motility rate, vitality, capacitation and acrosome reaction) from each rabbit buck were recorded.

The semen was collected twice a week, whereas blood samples were taken every two weeks. The semen samples were macroscopically evaluated, and only those displaying a homogenous white opalescent colour were included in the experiment, while those containing urine, cell debris, or lipid droplets were excluded from further analysis. At slaughtering the liver and testis of the animals were collected (Figure 1).

## Semen and blood sampling

The semen was collected with an artificial vagina maintained at temperature of 37°C, and stored in glass tubes to collect the semen of each rabbit (n = 270 semen samples/groups excluding the discarded ones). Then the semen was stored in sterile eppendorfs and immediately transported to the laboratory of the Department of Agricultural, Environmental and Food Science, University of Perugia.

Two ml of blood (one for serum and one for plasma) were taken from the auricular marginal ear vein, after the local application of an anaesthetic cream (EMLA®), using a syringe (2.5 mL) fitted with a butterfly needle. Serum was obtained from coagulated blood samples (at room temperature for 2 h). Plasma obtained from blood samples was collected into Na₂-EDTA tubes and immediately centrifuged at 2,500 × g for 15 mins at 4°C.

## Semen quality evaluation

Semen parameters such as colour, volume, vitality, capacitation and acrosome reaction, sperm concentration and motility were evaluated. Semen samples with anomalous colour (yellow, brownish) were discarded (n = 11 semen samples in total) as previously reported.

The sperm vitality was evaluated with the eosin test, the volume was evaluated using a graduated glass tube and sperm concentration was assessed with a Thoma-Zeiss cell counting chamber (BRAND, Wertheim, Germany) with an optical microscope (magnification x 20, Olympus CH-2; Olympus Optical Co., Ltd, Tokyo, Japan).

The sperm kinetics were analysed with a computer-based sperm analyser (CASA; model ISAS, Proiser, Valencia, Spain). The semen was diluted 1:30 with Tyrode Albumin Lactate Pyruvate (TALP) buffer, and the acquisition frequency was set to 100 HZ (Lattaioli and Castellini 1998) by a negative phase contrast optical system (Olympus CH-2) equipped with a CCD Sony camera.
<figure><img src="image_17.png" /><figcaption>**Figure 1.** Experimental protocol.</figcaption></figure>

For each sample, two drops and six microscopic fields were analysed for a total of 300 spermatozoa. The main parameters analysed were:

* Motility (%), defined as the number of mobile spermatozoa on the total number of spermatozoa present in the optic field;

* Curvilinear velocity (VCL, µm/sec), defined as the sum of the distances moved in each frame along the sampled path divided by the time taken for the spermatozoa to cover the track;

* Linearity (LIN, %), defined as the percentage of the VSL/VCL ratio (VSL, straight-line velocity);

* Amplitude of lateral head displacement (ALH, µm), defined as lateral displacement of the sperm head around its mean path.

For the assessment of sperm acrosome status and capacitation, the chlortetracycline (CTC) fluorescence test was performed as reported by Cocchia et al. (2011). Briefly, 55 µL of semen suspension were taken and inserted into eppendorf, to which 45 µL of CTC and 1 mg/mL of propide iodide were added. To fix the cells on slides, 8 µL of 12.5% parafolmaldeyde medium and a drop of 1,4-diazabicyclo[2.2.2]octane dissolved in phosphate-buffered saline (PBS) were added to ensure the maintenance of fluorescence. The sample was analysed with an epifluorescence microscope (OLYMPUS 335-425 CH2 excitation filter at 488 nm and 560 nm for the detection of propidium iodide and

CTC, respectively). Three different fluorescence models were detected: whole-head fluorescence of the sperm (not capacitated-NC), non-fluorescent band in the post-acrosomic region of the sperm head (capacitation-CP) or absence of fluorescence on the sperm head (acrosomal reaction-AR). A minimum of 300 cells was counted on each sample. Two replicate slides were counted for all semen samples, to ensure analytical repeatability.

For measuring the sperm responsiveness to dietary modification, the CP/NC ratio and the AR/CP ratio was also evaluated (Castellini et al. 2013).

## Sampling of rabbit organs

At the end of the experiment, the rabbits were euthanised after an overdose of barbiturates as approved by the Animal Ethics Monitoring Committee previously reported. The testis and liver were accurately removed, and placed in sterile tubes, immediately snap-frozen using liquid nitrogen and stored at -80°C for following analysis.

## Analytical determination on sperm, blood, and tissues

Lipid oxidation in seminal plasma (n = 30/group) and blood plasma (n = 15/group) (0.2 and 0.5 mL, respectively) was assessed using reactive thiobarbituric substances (TBARS) by measuring malondialdehyde (MDA)
<figure><img src="image_18.png" /></figure>

together with other substances that are reactive to 2-thiobarbituric acid (TBA), as reported by Buege and Aust (1978). Results were expressed in nmol MDA/mL.

The extraction of lipids from raw semen (n = 30/group), serum (n = 15/group), and organs (n = 15/group; liver and testis, about 5 g) was carried out according to the method described by Folch et al. (1957) and the esterification was carried out by the procedure of Christie (1982). The transmethylation procedure was carried out using as internal standard heneicosanoic acid methyl esters of (Sigma-Aldrich, Steinheim, Germany). The acid profile was determined using a Varian gas chromatograph (CP-3800) equipped with a flame ionisation detector (FID) and a capillary film column (Supelco, Bellefonte, PA, USA). The carrier gas was helium with a flow of 0.6 mL/min. The split ratio was set at 1:20. Identification of individual fatty acid methyl ester (FAME) by comparing the relative retention times of peaks in the sample with those of a standard mixture (FAME Mix Supelco; C4:0 to C24:0) plus cis-9 cis-12 C18:2; cis-9 cis-12-15 C18:3 and cis-9-12 C18:3 (all from Sigma-Aldrich). The fatty acids (FA) were expressed as a % of total FA. The average quantity of each FA was used to calculate the sum of total SFA, monounsaturated fatty acids (MUFA), n-3 and n-6 PUFA. The LC-PUFA included EPA, DPA and DHA for n-3 and ARA and DPA for n-6.

Several indices were used to estimate the desaturase and elongase activity of the analysed tissues as specified in Dal Bosco et al. (2012). In particular was calculated the activity of Δ9-desaturase (for both palmitic C16:0 and C18:0), elongase (Okada et al. 2005), thioesterase and both Δ5-desaturase and Δ6-desaturase enzymes (Dal Bosco et al. 2012), as reported:

$$ \Delta 9 - \text{desaturase (C18 : 1)} \\ = ((C18 : 1n - 9)/(C18 : 0 + C18 : 1n - 9)) \times 100 $$

$$ \Delta 9 - \text{desaturase}(C16 : 1 + C18 : 1) \\ = ((C16 : 1n - 7 + C18 : 1n - 9)/(C16 : 0 + C18 : 0 + C16 : 1n - 7 + C18 : 1n - 9)) \times 100 $$

$$ \text{Elongase} = ((C18 : 0)/(C16 : 0)) \times 100 $$

$$ \text{Thioesterase} = ((C16 : 0)/(C14 : 0)) \times 100 $$

$$ \Delta 5/\Delta 6 - \text{desaturase} \\ = [(C20 : 2n - 6 + C20 : 4n - 6 + EPA + C22 : 5n - 3 + DHA)/(LA + ALA + C20 : 2n - 6 + C20 : 4n - 6 + EPA + C22 : 5n - 3 + DHA)] \times 100. $$

The level of tocols (α-tocopherol and its isoforms γ and δ, and α and γ-tocotrienol) was measured

according to Schüep and Rettenmaier (1994) in both semen (n = 30/group) and blood plasma (n = 15/group) samples. Briefly, 0.2 mL of raw semen or blood plasma was mixed with 1 mL of water and 4 mL of an ethanol solution of 0.06% butylated hydroxytoluene (BHT) as synthetic antioxidants. The mixture was saponified with water/potassium hydroxide (KOH, 60%) at 70°C for 30 min, extracted with hexane/ethyl acetate (9/1, v/v) and centrifuged (2,000 x g for 10 min at 4°C). Two mL of the supernatant was transferred into a glass tube, dried under Nitrogen (N₂), and resuspended in 200 µL of acetonitrile. The pellet was re-extracted twice. Finally, 50 µL of filtrate was injected into the HPLC/FD (Hitachi Primade, including cooling autosampler 1,210, pump 1,110, fluorimetric detector 1,440 and diode array detector 1,430 and a Synergi Hydro-RP column, 4 µm, 4.6 × 100 mm; Phenomenex, Bologna, Italy). The flow rate was set at 2 mL/min. All tocopherols and tocotrienols were identified using a fluorimetric detector (excitation and emission wavelengths of 295 and 328 nm, respectively) and quantified using external calibration curves prepared with increasing amounts of pure standard solutions (Sigma-Aldrich, Bornem, Belgium) in ethanol. The limit of detection (LOD), determined as three times the signal-to-noise ratio, and limit of quantitation (LOQ), determined as ten times the signal-to-noise ratio, of tocols were also calculated. The LOD values (µg/ml) were γ-tocotrienol 0.022 α-tocotrienol 0.018, δ-Tocopherol 0.012, γ-Tocopherol 0.020, α-Tocopherol 0.015. The LOQ values (µg/ml) were γ-tocotrienol 0.052, α-tocotrienol 0.047, δ-Tocopherol 0.045, γ-Tocopherol 0.060, α-Tocopherol 0.055. Only the sum of tocols was used for statistical analysis.

Cholesterol was extracted from rabbit semen (n = 30/group; 200 µL) using the method of Folch et al. (1957) adapted for semen analysis by Mourvaki et al. (2010). Using a mixture of extraction composed by chloroform:methanol (2:1, v/v) and 0.6% NaCl. The chloroform extracts were pooled and dried under a flow of nitrogen. The residue was dissolved in 200 µL isopropanol and filtered through the regenerated cellulose syringe disposals (RC Phenex 4 mm, 0.26 µm pore size; Phenomenex SRL., Bologna, Italy) prior to injection into the same HPLC system previously described. The detector used was a diode previously reported, set at 210 nm. The quantification was done using pure standard (Sigma-Aldrich, Steinheim, Germany) in isopropanol and external calibration curve. The LOD cholesterol values was 0.010 µg/mL, whereas the LOQ values 0.030 µg/mL.
## Statistical analysis

Skewness and kurtosis of the traits analysed was evaluated to check the normality and the homogenous distribution of variance.

A mixed linear model considering the animal as random (proc repeated) was used to evaluate the effect of diet on lipid oxidation, fatty acid profile, and kinetics parameters of semen (STATA, 2015). Fatty acids profile of diets and organs (liver and testis) was evaluated on with a one-way model (fixed effect of diet). The estimated range of variation was intra-assay and inter-assay were less than 5% and 10%, respectively.

In tables are reported the least squares (LS) and the root mean square error (RMSE).

To define the significance of differences, we used the Turkey post-hoc test with a significance set at 0.05. The Pearson correlation coefficient between cholesterol, CP, and AR was also calculated.

## Results and discussion

Body of literature reported that PUFA n-3 dietary supplementation positively affects rabbit female efficiency (Quattrone et al. 2024; Rodríguez et al. 2019) and male reproductive traits as sperm motility and track speed (Castellini et al. 2019) as well as the fatty acid profile of semen (Mourvaki et al. 2010). Until now, the most effective source of PUFA n-3 has been found in extruded linseed (from 8 to 10%; Rodríguez et al. 2019) combined with antioxidants (Mattioli et al. 2022), which, however, may also lead to some technological issues in feed production and its economic sustainability.

In the present trial, we investigated an innovative strategy using *Padina pavonica* algae extract as an enhancer of n-3 fatty acids metabolism, to reduce the level of dietary linseed (≤ 5%).

In Table 2, the fatty acids profile of the experimental diets was reported. As expected, the diets differed mainly for the two essential polyunsaturated fatty acids and in particular for the greater presence of LA in the C diet and ALA (more than three times) in L and LPP diets, depending on its content in linseed. The incorporation of *Padina pavonica* algae in such a small quantity (0.2%) did not induce substantial changes except for traces of EPA and DHA in the LPP diet, as a result of the relative content in the algae extract (Pham et al. 2020).

In Table 3, the main traits of rabbit semen are reported. The dietary administration of linseed alone or with *Padina pavonica* addition did not affect the

<table><caption>Table 2. Fatty acid profile (% of total fatty acids) of the experimental diets.</caption><thead><tr><th>Fatty acids (%)</th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th></tr></thead><tbody><tr><td>C14:0</td><td>0.18</td><td>0.20</td><td>0.20</td><td>0.01</td></tr><tr><td>C15:0</td><td>0.09</td><td>0.08</td><td>0.08</td><td>0.01</td></tr><tr><td>C16:0</td><td>13.92</td><td>11.14</td><td>11.16</td><td>1.10</td></tr><tr><td>C16:1cis9</td><td>0.18</td><td>0.20</td><td>0.18</td><td>0.20</td></tr><tr><td>C17:0</td><td>0.12</td><td>0.11</td><td>0.10</td><td>0.01</td></tr><tr><td>C17:1</td><td>0.05</td><td>0.05</td><td>0.05</td><td>0.01</td></tr><tr><td>C18:0</td><td>3.07</td><td>3.82</td><td>3.48</td><td>0.02</td></tr><tr><td>C18:1</td><td>25.39</td><td>23.89</td><td>23.98</td><td>2.10</td></tr><tr><td>C18:2n-6 (LA)</td><td>47.63<sup>b</sup></td><td>32.41<sup>a</sup></td><td>33.53<sup>a</sup></td><td>4.23</td></tr><tr><td>C20:0</td><td>0.28</td><td>0.22</td><td>0.22</td><td>0.02</td></tr><tr><td>C18:3n-6</td><td>0.21</td><td>0.12</td><td>0.19</td><td>0.01</td></tr><tr><td>C18:3n-3 (ALA)</td><td>6.53<sup>a</sup></td><td>23.25<sup>b</sup></td><td>22.63<sup>b</sup></td><td>1.10</td></tr><tr><td>C20:2</td><td>n.d.</td><td>n.d.</td><td>0.04</td><td>0.01</td></tr><tr><td>C20:3n-3</td><td>n.d.</td><td>n.d.</td><td>0.02</td><td>0.01</td></tr><tr><td>C22:0</td><td>n.d.</td><td>n.d.</td><td>0.23</td><td>0.02</td></tr><tr><td>C22:1</td><td>n.d.</td><td>n.d.</td><td>0.04</td><td>0.03</td></tr><tr><td>C20:4n-6 (AA)</td><td>n.d.</td><td>n.d.</td><td>0.08</td><td>0.02</td></tr><tr><td>C20:5n-3 (EPA)</td><td>n.d.</td><td>n.d.</td><td>0.13</td><td>0.04</td></tr><tr><td>C24:1</td><td>n.d.</td><td>n.d.</td><td>0.03</td><td>0.03</td></tr><tr><td>C22:5n-3 (DPA)</td><td>n.d.</td><td>n.d.</td><td>0.05</td><td>0.04</td></tr><tr><td>C22:6n-3 (DHA)</td><td>n.d.</td><td>n.d.</td><td>0.06</td><td>0.01</td></tr></tbody></table>

C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed +0.2% *Padina Pavonica* algae; RMSE: Root mean standard error
LA: linoleic acid; ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid.
Each mean is the result of three analytical determinations. n.d.: not detectable.

On the same row different letters (a,b) means *p* < 0.05 differences.

libido, semen volume, live and dead cells, static sperm, non-progressive motility, curvilinear velocity (VCL), and oscillation index (WOB).

On the contrary, significant differences regarding sperm concentration, progressive motility, linearity (LIN), and the movement of the sperm head (ALH) were observed, and in particular, they were higher in both L and LPP with respect to the C group.

Higher values of progressive motile sperm and LIN might suggest a better fertilisation activity of the L and LPP sperm; however, the higher value of BCF in the L group, could also indicate a precapacitative-like status of sperm.

The capacitation is a status of sperm hyperactivation that precedes the acrosome reaction; when an ejaculated sperm shows a higher number of capacitated sperm, a reduction in fertility is generally observed, because many sperms are not able to fertilise the egg (Castellini et al., 2024).

The CP/NC ratio was the same in the different dietary groups, whereas AR/CP was higher in both L and LPP than control (Table 3) as already suggested by BCF values. Accordingly, it seems that the susceptibility of sperm to acrosome reaction was enhanced by the linseed and algae administration.

The changes in the different sperm sensitivity to AR can be traced back to a modified asset of lipid profile, mainly referred to the ALA proportion (Table 4) of the membranes (Mourvaki et al. 2010), and at the same
<figure><img src="image_19.png" /><figcaption>**Table 3.** Semen quality evaluations of rabbit bucks fed with experimental diets.</figcaption></figure>

<table><thead><tr><th>Parameters</th><th>Unit</th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th><th>p</th></tr></thead><tbody><tr><td><i>Libido</i></td><td>(Sec)</td><td>35</td><td>40</td><td>38</td><td>3.25</td><td>0.283</td></tr><tr><td>Sperm concentration</td><td>x 10<sup>6</sup>/mL</td><td>509.36<sup>a</sup></td><td>576.05<sup>b</sup></td><td>595.08<sup>b</sup></td><td>3.93</td><td>0.046</td></tr><tr><td>Live cells</td><td>%</td><td>78.21</td><td>80.00</td><td>78.70</td><td>1.06</td><td>0.686</td></tr><tr><td>Dead cells</td><td>"</td><td>21.78</td><td>20.00</td><td>21.29</td><td>1.06</td><td>0.686</td></tr><tr><td>NC</td><td>"</td><td>75.0</td><td>72.5</td><td>81.5</td><td>1.29</td><td>0.134</td></tr><tr><td>CP</td><td>"</td><td>3.0</td><td>2.0</td><td>1.0</td><td>1.02</td><td>0.217</td></tr><tr><td>AR</td><td>"</td><td>22.0</td><td>25.5</td><td>17.5</td><td>1.06</td><td>0.177</td></tr><tr><td>CP/NC</td><td></td><td>4.00</td><td>2.75</td><td>1.22</td><td>0.05</td><td>0.058</td></tr><tr><td>AR/CP</td><td></td><td>7.33<sup>a</sup></td><td>12.75<sup>b</sup></td><td>17.50<sup>b</sup></td><td>0.85</td><td>0.012</td></tr><tr><td>Static cells</td><td>"</td><td>25.00</td><td>18.85</td><td>20.00</td><td>0.11</td><td>0.087</td></tr><tr><td>Motile non progressive</td><td>"</td><td>64.00</td><td>60.00</td><td>63.00</td><td>0.11</td><td>0.384</td></tr><tr><td>Motile progressive</td><td>"</td><td>11.00<sup>a</sup></td><td>20.00<sup>b</sup></td><td>17.00<sup>ab</sup></td><td>0.10</td><td>0.001</td></tr><tr><td>VCL</td><td>µm/s</td><td>239.10</td><td>242.16</td><td>230.98</td><td>1.93</td><td>0.724</td></tr><tr><td>LIN</td><td>%</td><td>19.83<sup>a</sup></td><td>24.80<sup>b</sup></td><td>21.81<sup>ab</sup></td><td>0.67</td><td>&lt;0.001</td></tr><tr><td>WOB</td><td>%</td><td>47.70</td><td>47.39</td><td>49.06</td><td>0.64</td><td>0.118</td></tr><tr><td>ALH</td><td>µm</td><td>3.22<sup>ab</sup></td><td>3.47<sup>b</sup></td><td>3.32<sup>a</sup></td><td>0.24</td><td>0.013</td></tr><tr><td>BCF</td><td>Hz</td><td>16.39<sup>a</sup></td><td>25.34<sup>b</sup></td><td>19.52<sup>ab</sup></td><td>0.85</td><td>&lt;0.001</td></tr></tbody></table>

N = 270 semen samples/group. C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed +0.2% *Padina Pavonica* algae; RMSE: Root mean standard error; NC: non capacitated sperm; CP: capacitated sperm; AR: acrosome reacted sperm; VCL: Curvilinear velocity; LIN: Linear trajectory; WOB: Oscillation index; ALH: Movement of the sperm head; BCG: Flagellum beat cross frequency.

On the same row different letters (a,b) means *p* < 0.05 differences.

time, the flexibility and motility of sperm. In this complex picture also the cholesterol amount of semen and sperm membrane may also play an important role, although the seminal cholesterol was similar (*p* = 0.070) in the different groups. Indeed, the cholesterol and AR/CP were negatively correlated (r = -0.507), corroborating this hypothesis (Table 5).

Mourvaki et al. (2010) assessed an inverse correlation between the amount of seminal cholesterol and sperm AR in rabbit. Accordingly, Castellini et al. (2003) concluded that rabbits supplied with high dietary PUFA had sperm more susceptible to oxidation and undergo to an early acrosome reaction.

Changes in sperm membrane permeability are correlated to the shift in PUFA profile that occurred during sperm capacitation, and the cholesterol efflux from the sperm cells is considered the initial mandatory step (Cross 2004).

As previously shown, the fatty acid profile of sperm evidenced significant differences concerning the dietary supplementation. The main differences were attributable to the linseed without noticing substantial effects related to the addition of algae. In particular, the control group showed a significant increase of DPA n-6 reflecting the high dietary content of LA; at the same time, a reduction of ALA, DHA, PUFA n-3, and SFA content was observed.

Retterstøl et al. (1998) confirmed that Sertoli and testicular cells can convert LA and ALA into LC-PUFA (e.g. EPA, DPA, and DHA) due to the activity of Δ5- and Δ6-desaturase and elongase in these cells

<table><caption>**Table 4.** Seminal cholesterol (nmol/mL) and fatty acid profile (% total fatty acids) of rabbit sperm after dietary administration.</caption><thead><tr><th></th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th><th>p</th></tr></thead><tbody><tr><td>Cholesterol</td><td>326.56</td><td>311.94</td><td>288.43</td><td>5.810</td><td>0.070</td></tr><tr><td>C14:0</td><td>11.02</td><td>10.39</td><td>9.05</td><td>0.595</td><td>0.099</td></tr><tr><td>C16:0</td><td>18.89<sup>a</sup></td><td>20.87<sup>b</sup></td><td>21.85<sup>c</sup></td><td>0.226</td><td>0.045</td></tr><tr><td>C16:1</td><td>0.04</td><td>0.01</td><td>0.08</td><td>0.276</td><td>0.187</td></tr><tr><td>C17:0</td><td>0.13</td><td>0.22</td><td>0.19</td><td>0.204</td><td>0.740</td></tr><tr><td>C18:0</td><td>18.51</td><td>22.93</td><td>22.62</td><td>0.598</td><td>0.166</td></tr><tr><td>C18:1</td><td>15.78<sup>a</sup></td><td>16.93<sup>b</sup></td><td>16.96<sup>b</sup></td><td>0.920</td><td>0.044</td></tr><tr><td>C18:2n-6 (LA)</td><td>7.78<sup>b</sup></td><td>3.41<sup>a</sup></td><td>3.21<sup>a</sup></td><td>0.561</td><td>0.026</td></tr><tr><td>C18:3n-3 (ALA)</td><td>0.05<sup>a</sup></td><td>0.23<sup>b</sup></td><td>0.26<sup>b</sup></td><td>0.182</td><td>0.022</td></tr><tr><td>C20:3n-3</td><td>0.79</td><td>0.85</td><td>0.86</td><td>0.268</td><td>0.551</td></tr><tr><td>C20:4n-6 (AA)</td><td>1.16</td><td>1.12</td><td>0.88</td><td>0.259</td><td>0.236</td></tr><tr><td>C20:5n-3 (EPA)</td><td>0.78</td><td>0.58</td><td>0.59</td><td>0.270</td><td>0.180</td></tr><tr><td>C22:5n-6</td><td>23.81<sup>b</sup></td><td>19.67<sup>a</sup></td><td>19.74<sup>a</sup></td><td>0.976</td><td>0.049</td></tr><tr><td>C22:5n-3 (DPA)</td><td>2.03</td><td>1.99</td><td>2.30</td><td>0.360</td><td>0.291</td></tr><tr><td>C22:6n-3 (DHA)</td><td>0.67<sup>a</sup></td><td>1.22<sup>b</sup></td><td>1.27<sup>b</sup></td><td>0.043</td><td>0.062</td></tr><tr><td>SFA</td><td>47.64<sup>a</sup></td><td>54.41<sup>b</sup></td><td>53.71<sup>b</sup></td><td>0.735</td><td>0.019</td></tr><tr><td>MUFA</td><td>15.82<sup>a</sup></td><td>16.94<sup>b</sup></td><td>17.18<sup>c</sup></td><td>0.942</td><td>0.037</td></tr><tr><td>PUFA</td><td>36.99<sup>b</sup></td><td>28.85<sup>a</sup></td><td>28.9<sup>a</sup></td><td>1.073</td><td>0.033</td></tr><tr><td>PUFAn-3</td><td>4.32</td><td>4.77</td><td>5.27</td><td>0.484</td><td>0.124</td></tr><tr><td>PUFAn-6</td><td>32.67<sup>b</sup></td><td>24.18<sup>a</sup></td><td>23.83<sup>a</sup></td><td>1.003</td><td>0.037</td></tr></tbody></table>

N = 30/group. C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed +0.2% *Padina Pavonica* algae; RMSE: Root mean standard error; LA: linoleic acid; ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

On the same row different letters (a..c) means *p* < 0.05 differences.

<table><caption>**Table 5.** Pearson correlation coefficient between seminal cholesterol and capacitation ratios.</caption><thead><tr><th></th><th>Seminal cholesterol</th><th>CP/NC</th></tr></thead><tbody><tr><td>CP/NC</td><td>0.750**</td><td>-</td></tr><tr><td>AR/CP</td><td>-0.507*</td><td>-0.947*</td></tr></tbody></table>

NC: non capacitated sperm; CP: capacitated sperm; AR: acrosome reacted sperm.

(Mattioli et al. 2021). Rabbit, like rat, seems to preferentially incorporate LC-PUFA n-6 into the sperm membrane, and it was demonstrated that the linseed administration reduced the LA conversion because of the higher affinity of Δ6-desaturase for ALA (Sprecher 2002).

As stated above, spermatozoa of L and LPP groups have similar FA profiles, indicating a scarce stimulating effect of the *Padina pavonica* on fatty acid metabolism. At this regard, the body of literature showed a noticeable responsiveness of rabbit sperm to dietary manipulation. Previous experiments (supplementation of 10% extruded linseed; Castellini et al. 2019) showed a major effect on n-3 increase. The use of plant-based products such as extruded linseed (Curone et al. 2022) or goji berries (Menchetti et al. 2020; Agradi et al. 2021), improved the semen quality of rabbit bucks, such as the use of fish oil at a concentration from 2 to 3.5% (Rooke et al. 2001; Castellini et al. 2003; Kokoli et al. 2017).

<footnote>*p* < 0.05.</footnote>

<footnote>**p* < 0.01 for Diet effect.</footnote>
Furthermore, other dietary strategies, which included the use of algae mixed with natural antioxidants (i.e. polyphenols; Vizzari et al. 2021) or other plant extract (2% *Ulva lactuca* infiltration; Okab et al. 2013), demonstrated an improvement of reproductive parameters in rabbit bucks and does.

As occurred in sperm, the FA profile of blood (Table 6) differed mainly for the PUFA profile, with a significant increase in ALA and PUFA n-3 and a reduction of LA and PUFA n-6 in the two experimental groups (Table 4).

The oxidative status of blood and semen (Table 7) was better in the control diet. Despite L and LPP diets were supplemented with a higher level of α-tocopherol (250 mg/kg diets; Castellini et al. 2003), a higher TBARS in blood and semen was recorded, demonstrating that the amount of tocopherol was not enough to counterbalance the oxidative boost of linseed addition (higher TBARS). Different mixture of antioxidants or higher tocopherol amount should be tested (Mattioli et al. 2022).

Mattioli et al. (2021) also found a negative effect of linseed administration (10%) on lipid oxidation products (both TBARS and isoprostanes) of liver, testis and brain and low quantity of tocopherols when a standard level of vitamin E (50 mg/kg) was added in the rabbit diet.

Herein a higher seminal concentration of Vitamin E was recorded in both linseed-supplemented groups, probably, the additional dose of tocopheryl acetate is

essential but not sufficient to counterbalance the metabolic oxidative thrust.

Concerning the fatty acid composition of testis (Table 8) significant differences were observed in L and C groups. As detected in blood and sperm, also in testis, no significant effects could be attributed to the addition of algae extract compared to linseed alone. In particular, respect to the control, L and LPP showed an increase of ALA (p = 0.014) and DHA (p = 0.046) associated to a significant reduction of total PUFA (p = 0.041) and PUFA n-6 (LA and DPA, p = 0.005 and 0.004 respectively) due to the lower quantity of dietary LA. In these dietary conditions, the Δ6/Δ5-desaturase increased (p = 0.036), which should be interpreted as a predisposition of testis to synthesise n-3 fatty acids.

The results on the FA profile of the testes and sperm agree with those of Castellini et al. (2019), even if in that case the quantities of extruded linseed were higher and consequently the effects on n-3 were more evident.

As previously noted, linseed administration increased ALA with a contemporary increase in LC-PUFA, which confirms the ability of both liver and testis to elongate and desaturate ALA (Lin and Salem 2007) demonstrating the ability of the rabbit tissues to synthesise and accumulate LC-PUFA. The LC-PUFA accumulation observed in the sperm membrane of experimental diets contributes to the improvement of motion traits of cells, probably due to a change in membrane fluidity. Indeed, the track speed of the L and LPP groups increased by about 30%. As a result, the tracing of dietary PUFA in testes and sperm is particularly interesting.

The significant increase of DHA observed in the liver of the LLP rabbits (0.19, 0.28 and 0.40% respectively for C, L and LPP groups, *p* < 0.05; Table 9) agrees with the results of Tsukui et al. (2007) that observed in mice an increase in DHA and AA as a result of a dietary supplementation with fucoxanthin and fucoxanthinol due to a modification in the biosynthesis and degradation of

<table><caption>**Table 6.** Fatty acid profile (% total fatty acids) of rabbit blood after dietary administration.</caption><thead><tr><th></th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th><th>p</th></tr></thead><tbody><tr><td>C14:0</td><td>0.66</td><td>1.26</td><td>0.65</td><td>0.33</td><td>0.431</td></tr><tr><td>C16:0</td><td>17.78</td><td>16.84</td><td>16.9</td><td>0.57</td><td>0.065</td></tr><tr><td>C16:1</td><td>0.50</td><td>0.54</td><td>0.66</td><td>0.21</td><td>0.158</td></tr><tr><td>C17:0</td><td>0.83</td><td>0.73</td><td>0.82</td><td>0.22</td><td>0.119</td></tr><tr><td>C18:0</td><td>9.21</td><td>9.23</td><td>9.14</td><td>0.15</td><td>0.477</td></tr><tr><td>C18:1</td><td>10.64</td><td>10.85</td><td>11.57</td><td>0.49</td><td>0.911</td></tr><tr><td>C18:2n-6 (LA)</td><td>23.41<sup>b</sup></td><td>17.51<sup>a</sup></td><td>17.36<sup>a</sup></td><td>0.75</td><td>0.070</td></tr><tr><td>C18:3n-3 (ALA)</td><td>30.42<sup>a</sup></td><td>34.98<sup>b</sup></td><td>33.61<sup>b</sup></td><td>0.87</td><td>0.023</td></tr><tr><td>C20:3n-3 (EPA)</td><td>1.80<sup>a</sup></td><td>4.13<sup>b</sup></td><td>5.85<sup>b</sup></td><td>0.62</td><td>0.035</td></tr><tr><td>C20:4n-6 (AA)</td><td>3.02<sup>a</sup></td><td>1.55<sup>b</sup></td><td>1.57<sup>b</sup></td><td>0.33</td><td>0.239</td></tr><tr><td>C20:5n-3 (DPA)</td><td>0.85</td><td>0.82</td><td>0.78</td><td>0.30</td><td>0.330</td></tr><tr><td>C22:5n-6 (DPA)</td><td>0.20</td><td>0.12</td><td>0.14</td><td>0.14</td><td>0.421</td></tr><tr><td>C22:5n-3 (DHA)</td><td>0.61<sup>a</sup></td><td>1.52<sup>b</sup></td><td>1.51<sup>b</sup></td><td>0.26</td><td>0.326</td></tr><tr><td>SFA</td><td>28.48</td><td>28.06</td><td>27.51</td><td>0.67</td><td>0.214</td></tr><tr><td>MUFA</td><td>11.14</td><td>11.39</td><td>12.23</td><td>0.76</td><td>0.061</td></tr><tr><td>PUFA</td><td>60.29</td><td>60.63</td><td>60.82</td><td>0.90</td><td>0.081</td></tr><tr><td>PUFAn-3</td><td>33.67<sup>a</sup></td><td>41.45<sup>b</sup></td><td>41.75<sup>b</sup></td><td>0.61</td><td>0.019</td></tr><tr><td>PUFAn-6</td><td>26.62<sup>b</sup></td><td>19.18<sup>a</sup></td><td>19.07<sup>a</sup></td><td>0.89</td><td>0.034</td></tr></tbody></table>

N = 15/group. C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed + 0.2% *Padina Pavonica* algae; RMSE: Root mean standard error; LA: linoleic acid; ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

On the same row different letters (a,b) means *p* < 0.05 for Diet effect.

<table><caption>**Table 7.** Oxidative status (TBARS, nmol MDA/mL, and tocols content, nmol/mL) of rabbit semen and blood after dietary administration.</caption><thead><tr><th></th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th><th>p</th></tr></thead><tbody><tr><td>Semen</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TBARS</td><td>5.75<sup>a</sup></td><td>11.21<sup>b</sup></td><td>10.98<sup>b</sup></td><td>1.06</td><td>0.025</td></tr><tr><td>Tocols</td><td>1.31<sup>a</sup></td><td>2.39<sup>b</sup></td><td>1.85<sup>ab</sup></td><td>0.52</td><td>0.020</td></tr><tr><td>Blood</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TBARS</td><td>26.72<sup>A</sup></td><td>28.71<sup>B</sup></td><td>29.28<sup>B</sup></td><td>1.29</td><td>0.009</td></tr><tr><td>Tocols</td><td>0.35</td><td>0.47</td><td>0.63</td><td>0.28</td><td>0.279</td></tr></tbody></table>

N = 15/group. C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed + 0.2% *Padina Pavonica* algae; RMSE: Root mean standard error; TBARS: Thiobarbituric reactive substances; MDA: malondialdehyde.

On the same row different letters (a,b) means *p* < 0.05, capital letter *p* < 0.01.
<figure><img src="image_20.png" /><figcaption>**Table 8.** Main fatty acid (% total fatty acids) and estimated indices of rabbit testis.</figcaption></figure>

<table><thead><tr><th></th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th><th>p</th></tr></thead><tbody><tr><td>C14:0</td><td>0.96</td><td>0.89</td><td>0.91</td><td>0.195</td><td>0.999</td></tr><tr><td>C14:1</td><td>0.25</td><td>0.27</td><td>0.30</td><td>0.150</td><td>0.694</td></tr><tr><td>C16:0</td><td>19.94</td><td>20.12</td><td>19.24</td><td>0.945</td><td>0.423</td></tr><tr><td>C16:1</td><td>1.81<sup>a</sup></td><td>4.01<sup>b</sup></td><td>4.09<sup>b</sup></td><td>0.662</td><td>0.022</td></tr><tr><td>C17:0</td><td>0.54</td><td>0.42</td><td>0.52</td><td>0.169</td><td>0.196</td></tr><tr><td>C17:1</td><td>0.15</td><td>0.26</td><td>0.29</td><td>0.169</td><td>0.084</td></tr><tr><td>C18:0</td><td>6.86</td><td>6.56</td><td>7.14</td><td>0.797</td><td>0.176</td></tr><tr><td>C18:1</td><td>18.14</td><td>20.01</td><td>19.16</td><td>1.113</td><td>0.097</td></tr><tr><td>C18:2n-6 (LA)</td><td>23.08<sup>b</sup></td><td>20.25<sup>a</sup></td><td>21.71<sup>a</sup></td><td>1.343</td><td>0.041</td></tr><tr><td>C20:0</td><td>0.11</td><td>0.07</td><td>0.07</td><td>0.097</td><td>0.138</td></tr><tr><td>C18:3n-6</td><td>0.17</td><td>0.12</td><td>0.11</td><td>0.115</td><td>0.130</td></tr><tr><td>C18:3 n-3 (ALA)</td><td>1.79<sup>a</sup></td><td>4.44<sup>b</sup></td><td>5.81<sup>b</sup></td><td>0.823</td><td>0.014</td></tr><tr><td>CLAc9,t11</td><td>0.03</td><td>0.04</td><td>0.01</td><td>0.206</td><td>0.541</td></tr><tr><td>C20:2</td><td>0.37</td><td>0.32</td><td>0.36</td><td>0.120</td><td>0.547</td></tr><tr><td>C20:3n-6</td><td>4.58</td><td>3.25</td><td>2.93</td><td>0.676</td><td>0.087</td></tr><tr><td>C22:1</td><td>0.04</td><td>0.08</td><td>0.07</td><td>0.080</td><td>0.438</td></tr><tr><td>C20:4n-6 (AA)</td><td>6.48<sup>b</sup></td><td>4.28<sup>a</sup></td><td>4.48<sup>a</sup></td><td>0.802</td><td>0.034</td></tr><tr><td>C22:2</td><td>0.09</td><td>0.16</td><td>0.20</td><td>0.055</td><td>0.546</td></tr><tr><td>C24:0</td><td>0.03</td><td>0.02</td><td>0.02</td><td>0.122</td><td>0.073</td></tr><tr><td>C20:5n-3 (EPA)</td><td>0.16</td><td>0.11</td><td>0.08</td><td>0.057</td><td>0.688</td></tr><tr><td>C22:5n-3 (DPA)</td><td>0.02</td><td>0.03</td><td>0.03</td><td>0.421</td><td>0.120</td></tr><tr><td>C22:5n-6</td><td>5.95</td><td>5.08</td><td>4.96</td><td>0.891</td><td>0.071</td></tr><tr><td>C22:6n-3 (DHA)</td><td>0.17<sup>a</sup></td><td>0.83<sup>b</sup></td><td>0.84<sup>b</sup></td><td>0.147</td><td>0.046</td></tr><tr><td>others</td><td>7.05</td><td>6.94</td><td>6.03</td><td>0.779</td><td>0.444</td></tr><tr><td>SFA</td><td>28.46</td><td>28.1</td><td>27.89</td><td>1.205</td><td>0.021</td></tr><tr><td>MUFA</td><td>20.39</td><td>24.63</td><td>23.91</td><td>1.294</td><td>0.063</td></tr><tr><td>PUFA</td><td>44.78<sup>b</sup></td><td>40.45<sup>a</sup></td><td>42.35<sup>ab</sup></td><td>0.657</td><td>0.041</td></tr><tr><td>n-3</td><td>20.52</td><td>21.79</td><td>20.68</td><td>0.696</td><td>0.012</td></tr><tr><td>n-6</td><td>40.26<sup>b</sup></td><td>32.98<sup>a</sup></td><td>34.19<sup>a</sup></td><td>0.576</td><td>0.014</td></tr><tr><td>n-6/n-3</td><td>2.89</td><td>3.55</td><td>3.97</td><td>0.809</td><td>0.005</td></tr><tr><td>PUFA/SFA</td><td>1.57</td><td>1.44</td><td>1.52</td><td>0.292</td><td>0.039</td></tr><tr><td colspan="6"><em>Estimated lipid indexes</em></td></tr><tr><td>Elongase</td><td>0.34</td><td>0.33</td><td>0.37</td><td>0.148</td><td>0.629</td></tr><tr><td>Thioesterase</td><td>20.77</td><td>22.61</td><td>21.14</td><td>1.198</td><td>0.274</td></tr><tr><td>Δ9-desaturase (18)</td><td>72.56</td><td>75.31</td><td>72.85</td><td>1.724</td><td>0.115</td></tr><tr><td>Δ9-desaturase (16 + 18)</td><td>42.67</td><td>47.38</td><td>46.85</td><td>1.745</td><td>0.041</td></tr><tr><td>Δ5/Δ6-desaturase</td><td>43.28<sup>a</sup></td><td>37.49<sup>b</sup></td><td>34.25<sup>b</sup></td><td>0.954</td><td>0.036</td></tr></tbody></table>

N = 15/group. C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed +0.2% *Padina Pavonica* algae; RMSE: Root mean standard error; LA: linoleic acid; ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

On the same row different letters (a,b) means *p* < 0.05 for Diet effect.

PUFA in the liver. Furthermore, the increase in hepatic AA was less evident with respect to DHA, especially in mice fed fucoxanthin diets. Considering that Δ6-desaturase is known to be the rate-limiting enzyme in such pathways in mammals (Sprecher 1981; Cho et al. 1999) these Authors found that dietary carotenoids of brown algae binding C18:4n-3 and EPA may modify the biosynthesis and metabolic pathways of PUFA n-3 and n-6, with particular predisposition to DHA.

Finally, Pangestuti et al. (2022) highlighted a particular role of some brown algae, such as *Padina pavonica*, in the activation of lipid metabolism as well as a large amount of nutraceutical effects.

## Conclusions

The present study demonstrated a low effect of *Padina pavonica* extract added to extruded linseed

<table><caption>**Table 9.** Fatty acid profile (% total fatty acids) of rabbit liver.</caption><thead><tr><th></th><th>C</th><th>L</th><th>LPP</th><th>RMSE</th><th>p</th></tr></thead><tbody><tr><td>C14</td><td>0.21</td><td>0.23</td><td>0.21</td><td>0.173</td><td>0.247</td></tr><tr><td>C14:1</td><td>0.29</td><td>0.28</td><td>0.28</td><td>0.119</td><td>0.975</td></tr><tr><td>C16</td><td>15.32</td><td>15.27</td><td>15.08</td><td>0.852</td><td>0.024</td></tr><tr><td>C16:1</td><td>0.41</td><td>0.44</td><td>0.16</td><td>0.292</td><td>0.392</td></tr><tr><td>C17</td><td>1.04</td><td>0.9</td><td>1.15</td><td>0.230</td><td>0.147</td></tr><tr><td>C17:1</td><td>0.18</td><td>0.14</td><td>0.08</td><td>0.152</td><td>0.218</td></tr><tr><td>C18</td><td>20.49</td><td>22.62</td><td>22.69</td><td>0.954</td><td>0.171</td></tr><tr><td>C18:1</td><td>12.52</td><td>12.51</td><td>12.53</td><td>0.882</td><td>0.317</td></tr><tr><td>C18:2n-6 (LA)</td><td>34.47<sup>b</sup></td><td>30.69<sup>a</sup></td><td>30.82<sup>a</sup></td><td>0.584</td><td>0.005</td></tr><tr><td>C20</td><td>0.07</td><td>0.07</td><td>0.08</td><td>0.061</td><td>0.302</td></tr><tr><td>C18:3 n-6</td><td>0.1</td><td>0.1</td><td>0.12</td><td>0.107</td><td>0.776</td></tr><tr><td>C18:3 n-3 (ALA)</td><td>1.85<sup>a</sup></td><td>3.66<sup>b</sup></td><td>3.29<sup>b</sup></td><td>0.545</td><td>0.002</td></tr><tr><td>CLAc9,t11</td><td>0.07</td><td>0.02</td><td>0.02</td><td>0.113</td><td>0.237</td></tr><tr><td>C20:2</td><td>0.71</td><td>0.59</td><td>0.67</td><td>0.198</td><td>0.466</td></tr><tr><td>C20:3n-6</td><td>0.66</td><td>0.64</td><td>0.68</td><td>0.140</td><td>0.763</td></tr><tr><td>C22:1</td><td>0.02</td><td>0.01</td><td>0.02</td><td>0.042</td><td>0.560</td></tr><tr><td>C20:3n-3</td><td>0.09</td><td>0.16</td><td>0.20</td><td>0.137</td><td>0.115</td></tr><tr><td>C20:4n-6 (AA)</td><td>6.46</td><td>6.69</td><td>6.93</td><td>0.579</td><td>0.954</td></tr><tr><td>C22:2</td><td>0.02</td><td>0.03</td><td>0.03</td><td>0.054</td><td>0.326</td></tr><tr><td>C24</td><td>0.08</td><td>0.15</td><td>0.16</td><td>0.118</td><td>0.257</td></tr><tr><td>C20:5n-3 (EPA)</td><td>0.02</td><td>0.05</td><td>0.06</td><td>0.121</td><td>0.397</td></tr><tr><td>C22:5n-3 (DPA)</td><td>0.66</td><td>0.55</td><td>0.68</td><td>0.225</td><td>0.282</td></tr><tr><td>C22:5n-6</td><td>0.50<sup>b</sup></td><td>0.29<sup>a</sup></td><td>0.26<sup>a</sup></td><td>0.204</td><td>0.004</td></tr><tr><td>C22:6n-3 (DHA)</td><td>0.19<sup>a</sup></td><td>0.28<sup>b</sup></td><td>0.40<sup>c</sup></td><td>0.138</td><td>0.048</td></tr><tr><td>others</td><td>3.38</td><td>3.63</td><td>3.89</td><td>0.352</td><td>0.275</td></tr><tr><td>SFA</td><td>37.21</td><td>39.24</td><td>39.37</td><td>0.678</td><td>0.274</td></tr><tr><td>MUFA</td><td>13.42</td><td>13.38</td><td>13.07</td><td>0.938</td><td>0.321</td></tr><tr><td>PUFA</td><td>46.02</td><td>43.76</td><td>43.90</td><td>0.760</td><td>0.096</td></tr><tr><td>PUFAn-3</td><td>3.01</td><td>4.71</td><td>4.55</td><td>0.516</td><td>0.006</td></tr><tr><td>PUFAn-6</td><td>42.19</td><td>38.41</td><td>38.81</td><td>0.740</td><td>0.226</td></tr><tr><td>n-6/n-3</td><td>14.02<sup>b</sup></td><td>8.17<sup>a</sup></td><td>8.49<sup>a</sup></td><td>0.951</td><td>0.024</td></tr><tr><td>PUFA/SFA</td><td>1.24</td><td>1.12</td><td>1.12</td><td>0.132</td><td>0.210</td></tr><tr><td colspan="6"><em>Estimated lipid indexes</em></td></tr><tr><td>Elongase</td><td>1.34</td><td>1.48</td><td>1.50</td><td>0.371</td><td>0.068</td></tr><tr><td>Thioesterase</td><td>72.95</td><td>66.39</td><td>71.81</td><td>3.535</td><td>0.290</td></tr><tr><td>Δ9-desaturase (18)</td><td>37.93</td><td>35.61</td><td>35.58</td><td>1.511</td><td>0.236</td></tr><tr><td>Δ9-desaturase (16 + 18)</td><td>26.53</td><td>25.47</td><td>25.15</td><td>1.185</td><td>0.412</td></tr><tr><td>Δ5/Δ6-desaturase</td><td>19.42</td><td>20.09</td><td>20.69</td><td>0.794</td><td>0.655</td></tr></tbody></table>

N = 15/group. C: Control diet; L: standard diet + 5% linseed; LPP: standard diet + 5% linseed +0.2% *Padina Pavonica* algae; RMSE: Root mean standard error; LA: linoleic acid; ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

On the same row different letters (a,c) means *p* < 0.05.

diet on seminal characteristics. The expected synergistic or metabolic-enhancing effect of the algae extract in combination with linseed was not substantially confirmed under the current experimental conditions. Probably, the changes in FA of sperm and tissues are not sufficient to determine significant improvement in the kinetic and biological performance of the spermatozoa.

However, an improvement, attributable to linseed alone, compared to the control, was observed. Accordingly, it is not possible to draw any definitive conclusion on the interaction between algae extract and linseed because the expected activation of linseed-algae diets was only slightly observed.

Further studies should investigate other combinations of linseed and algae to find more effective interactions. Possibly, to obtain significant effects, it is
necessary to increase the quantity of algae extract and
try to better define their mechanisms of action.

## Acknowledgements

We thank the ASIC-Associazione Scientifica Italiana di
Coniglicoltura (WRSA-latlian Branch) for publishing support.

## Disclosure statement

No potential conflict of interest was reported by the
author(s)

## Funding

This research was funded by PRIMA project 'Omega rabbit: food for health benefit' (ORABBIT: G15F21000810001).

## Data availability statement

The data that support the findings of this study are available
from the corresponding author, SM, upon reasonable
request.

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